Comparison of Four Protocols for In Vitro Differentiation of Human Embryonic Stem Cells into Trophoblast Lineages by BMP4 and Dual Inhibition of Activin/Nodal and FGF2 Signaling.

Human embryonic stem cells (hESCs) cultured in media containing bone morphogenic protein 4 (BMP4; B) differentiate into trophoblast-like cells. Supplementing media with inhibitors of activin/nodal signaling (A83-01) and of fibroblast growth factor 2 (PD173074) suppresses mesoderm and endoderm formation and improves specification of trophoblast-like lineages, but with variable effectiveness. We compared differentiation in four BMP4-containing media: mTeSR1-BMP4 only, mTeSR1-BAP, basal medium with BAP (basal-BAP), and a newly defined medium, E7-BAP. These media variably drive early differentiation towards trophoblast-like lineages with upregulation of early trophoblast markers CDX2 and KRT7 and downregulation of pluripotency markers (OCT4 and NANOG). As expected, based on differences between media in FGF2 and its inhibitors, downregulation of mesendoderm marker EOMES was variable between media. By day 7, only hESCs grown in E7-BAP or basal-BAP expressed HLA-G protein, indicating the presence of cells with extravillous trophoblast characteristics. Expression of HLA-G and other differentiation markers (hCG, KRT7, and GCM1) was highest in basal-BAP, suggesting a faster differentiation in this medium, but those cultures were more inhomogeneous and still expressed some endodermal and pluripotency markers. In E7-BAP, HLA-G expression increased later and was lower. There was also a low but maintained expression of some C19MC miRNAs, with more CpG hypomethylation of the ELF5 promoter, suggesting that E7-BAP cultures differentiate slower along the trophoblast lineage. We conclude that while all protocols drive differentiation into trophoblast lineages with varying efficiency, they have advantages and disadvantages that must be considered when selecting a protocol for specific experiments.

Loss of the Maternal Effect Gene Nlrp2 Alters the Transcriptome of Ovulated Mouse Oocytes and Impacts Expression of Histone Demethylase KDM1B.

The subcortical maternal complex (SCMC) is a multiprotein complex in oocytes and preimplantation embryos that is encoded by maternal effect genes. The SCMC is essential for zygote-to-embryo transition, early embryogenesis, and critical zygotic cellular processes, including spindle positioning and symmetric division. Maternal deletion of Nlrp2, which encodes an SCMC protein, results in increased early embryonic loss and abnormal DNA methylation in embryos. We performed RNA sequencing on pools of meiosis II (MII) oocytes from wild-type and Nlrp2-null female mice that were isolated from cumulus-oocyte complexes (COCs) after ovarian stimulation. Using a mouse reference genome-based analysis, we found 231 differentially expressed genes (DEGs) in Nlrp2-null compared to WT oocytes (123 up- and 108 downregulated; adjusted p < 0.05). The upregulated genes include Kdm1b, a H3K4 histone demethylase required during oocyte development for the establishment of DNA methylation marks at CpG islands, including those at imprinted genes. The identified DEGs are enriched for processes involved in neurogenesis, gland morphogenesis, and protein metabolism and for post-translationally methylated proteins. When we compared our RNA sequencing data to an oocyte-specific reference transcriptome that contains many previously unannotated transcripts, we found 228 DEGs, including genes not identified with the first analysis. Interestingly, 68% and 56% of DEGs from the first and second analyses, respectively, overlap with oocyte-specific hyper- and hypomethylated domains. This study shows that there are substantial changes in the transcriptome of mouse MII oocytes from female mice with loss of function of Nlrp2, a maternal effect gene that encodes a member of the SCMC.

Take your mother's ferry: preimplantation embryo development requires maternal karyopherins for nuclear transport.

The genetic basis of preimplantation embryo arrest is slowly being unraveled. Recent discoveries point to maternally expressed proteins required for cellular functions before the embryonic genome is activated. In this issue of the JCI, Wang, Miyamoto, et al. suggest a critical role for karyopherin-mediated protein cargo transport between oocyte cytoplasm and nucleus. Defective maternal oocyte-expressed human karyopherin subunit α7 (KPNA7) and mouse KPNA2 fail to bind a critical substrate, ribosomal L1 domain-containing protein 1 (RSL1D1), affecting its transport to the nucleus. As shown in embryos of Kpna2-null females, the consequences are disrupted zygotic genome activation and arrest of development. These findings have important implications for diagnosis and treatment of female infertility.

Hyperactivation is sufficient to release porcine sperm from immobilized oviduct glycans.

Fertilizing sperm are retained by adhesion to specific glycans on the epithelium of the oviduct forming a reservoir before sperm are released from the reservoir so fertilization can ensue. Capacitated sperm lose affinity for the oviduct epithelium but the components of capacitation that are important for sperm release are uncertain. One important correlate of capacitation is the development of hyperactivated motility. Hyperactivation is characterized by asymmetrical flagellar beating with high beat amplitude. We tested whether the development of full-type asymmetrical motility was sufficient to release sperm from immobilized oviduct glycans. Sperm hyperactivation was induced by four different compounds, a cell-permeable cAMP analog (cBiMPS), CatSper activators (4-aminopyridine and procaine), and an endogenous steroid (progesterone). Using standard analysis (CASA) and direct visualization with high-speed video microscopy, we first confirmed that all four compounds induced hyperactivation. Subsequently, sperm were allowed to bind to immobilized oviduct glycans, and compounds or vehicle controls were added. All compounds caused sperm release from immobilized glycans, demonstrating that hyperactivation was sufficient to release sperm from oviduct cells and immobilized glycans. Pharmacological inhibition of the non-genomic progesterone receptor and CatSper diminished sperm release from oviduct glycans. Inhibition of the proteolytic activities of the ubiquitin-proteasome system (UPS), implicated in the regulation of sperm capacitation, diminished sperm release in response to all hyperactivation inducers. In summary, induction of sperm hyperactivation was sufficient to induce sperm release from immobilized oviduct glycans and release was dependent on CatSper and the UPS.

DNA Methylation Dynamics in the Female Germline and Maternal-Effect Mutations That Disrupt Genomic Imprinting.

Genomic imprinting is an epigenetic marking process that results in the monoallelic expression of a subset of genes. Many of these 'imprinted' genes in mice and humans are involved in embryonic and extraembryonic growth and development, and some have life-long impacts on metabolism. During mammalian development, the genome undergoes waves of (re)programming of DNA methylation and other epigenetic marks. Disturbances in these events can cause imprinting disorders and compromise development. Multi-locus imprinting disturbance (MLID) is a condition by which imprinting defects touch more than one locus. Although most cases with MLID present with clinical features characteristic of one imprinting disorder. Imprinting defects also occur in 'molar' pregnancies-which are characterized by highly compromised embryonic development-and in other forms of reproductive compromise presenting clinically as infertility or early pregnancy loss. Pathogenic variants in some of the genes encoding proteins of the subcortical maternal complex (SCMC), a multi-protein complex in the mammalian oocyte, are responsible for a rare subgroup of moles, biparental complete hydatidiform mole (BiCHM), and other adverse reproductive outcomes which have been associated with altered imprinting status of the oocyte, embryo and/or placenta. The finding that defects in a cytoplasmic protein complex could have severe impacts on genomic methylation at critical times in gamete or early embryo development has wider implications beyond these relatively rare disorders. It signifies a potential for adverse maternal physiology, nutrition, or assisted reproduction to cause epigenetic defects at imprinted or other genes. Here, we review key milestones in DNA methylation patterning in the female germline and the embryo focusing on humans. We provide an overview of recent findings regarding DNA methylation deficits causing BiCHM, MLID, and early embryonic arrest. We also summarize identified SCMC mutations with regard to early embryonic arrest, BiCHM, and MLID.

Progesterone induces porcine sperm release from oviduct glycans in a proteasome-dependent manner.

In mammals, the oviduct retains sperm, forming a reservoir from which they are released in synchrony with ovulation. However, the mechanisms underlying sperm release are unclear. Herein, we first examined in greater detail the release of sperm from the oviduct reservoir by sex steroids, and secondly, if the ubiquitin-proteasome system (UPS) mediates this release in vitro. Sperm were allowed to bind to oviductal cells or immobilized oviduct glycans, either bi-SiaLN or a suLeX, and channeled with steroids in the presence or absence of proteasome inhibitors. Previously, we have demonstrated progesterone-induced sperm release from oviduct cells and immobilized glycans in a steroid-specific manner. Herein, we found that the release of sperm from an immobilized oviduct glycan, a six-sialylated branched structure, and from immobilized fibronectin was inhibited by the CatSper blocker NNC 055-0396, akin to the previously reported ability of NNC 055-0396 to inhibit sperm release from another oviduct glycan, sulfated Lewis-X trisaccharide. Thus, CatSper may be required for release of sperm from a variety of adhesion systems. One possible mechanism for sperm release is that glycan receptors on sperm are degraded by proteasomes or shed from the sperm surface by proteasomal degradation. Accordingly, the inhibition of proteasomal degradation blocked sperm release from oviduct cell aggregates both immobilized oviduct glycans as well as fibronectin. In summary, progesterone-induced sperm release requires both active CatSper channels and proteasomal degradation, suggesting that hyperactivation and proteolysis are vital parts of the mechanism by which sperm move from the oviduct reservoir to the site of fertilization.

Reproductive Outcomes from Maternal Loss of Nlrp2 Are Not Improved by IVF or Embryo Transfer Consistent with Oocyte-Specific Defect.

Nlrp2 encodes a protein of the oocyte subcortical maternal complex (SCMC), required for embryo development. We previously showed that loss of maternal Nlrp2 in mice causes subfertility, smaller litters with birth defects, and growth abnormalities in offspring, indicating that Nlrp2 is a maternal effect gene and that all embryos from Nlrp2-deficient females that were cultured in vitro arrested before the blastocysts stage. Here, we used time-lapse microscopy to examine the development of cultured embryos from superovulated Nlrp2-deficient and wild-type mice after in vivo and in vitro fertilization. Embryos from Nlrp2-deficient females had similar abnormal cleavage and fragmentation and arrested by blastocyst stage, irrespective of fertilization mode. This indicates that in vitro fertilization does not further perturb or improve the development of cultured embryos. We also transferred embryos from superovulated Nlrp2-deficient and wild-type females to wild-type recipients to investigate if the abnormal reproductive outcomes of Nlrp2-deficient females are primarily driven by oocyte dysfunction or if a suboptimal intra-uterine milieu is a necessary factor. Pregnancies with transferred embryos from Nlrp2-deficient females produced smaller litters, stillbirths, and offspring with birth defects and growth abnormalities. This indicates that the reproductive phenotype is oocyte-specific and is not rescued by development in a wild-type uterus. We further found abnormal DNA methylation at two maternally imprinted loci in the kidney of surviving young adult offspring, confirming persistent DNA methylation disturbances in surviving offspring. These findings have implications for fertility treatments for women with mutations in NLRP2 and other genes encoding SCMC proteins.

Adhesion to oviduct glycans regulates porcine sperm Ca2+ influx and viability.

Before fertilization, sperm bind to epithelial cells of the oviduct isthmus to form a reservoir that regulates sperm viability and capacitation. The sperm reservoir maintains optimum fertility in species, like swine, in which semen deposition and ovulation may not be well synchronized. We demonstrated previously that porcine sperm bind to two oviductal glycan motifs, a biantennary 6-sialylated N-acetyllactosamine (bi-SiaLN) oligosaccharide and 3-O-sulfated Lewis X trisaccharide (suLeX). Here, we assessed the ability of these glycans to regulate sperm Ca2+ influx, capacitation and affect sperm lifespan. After 24 h, the viability of sperm bound to immobilized bi-SiaLN and suLeX was higher (46% and 41% respectively) compared to viability of free-swimming sperm (10-12%). Ca2+ is a central regulator of sperm function so we assessed whether oviduct glycans could affect the Ca2+ influx that occurs during capacitation. Using a fluorescent intracellular Ca2+ probe, we observed that both oviduct glycans suppressed the Ca2+ increase that occurs during capacitation. Thus, specific oviduct glycans can regulate intracellular Ca2+. Because the increase in intracellular Ca2+ was suppressed by oviduct glycans, we examined whether glycans affected capacitation, as determined by protein tyrosine phosphorylation and the ability to undergo a Ca2+ ionophore-induced acrosome reaction. We found no discernable suppression of capacitation in sperm bound to oviduct glycans. We also detected no effect of oviduct glycans on sperm motility during capacitation. In summary, LeX and bi-SiaLN glycan motifs found on oviduct oligosaccharides suppress the Ca2+ influx that occurs during capacitation and extend sperm lifespan but do not affect sperm capacitation or motility.

Sperm Cohort-Specific Zinc Signature Acquisition and Capacitation-Induced Zinc Flux Regulate Sperm-Oviduct and Sperm-Zona Pellucida Interactions.

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.

Release of Porcine Sperm from Oviduct Cells is Stimulated by Progesterone and Requires CatSper.

Sperm storage in the female reproductive tract after mating and before ovulation is a reproductive strategy used by many species. When insemination and ovulation are poorly synchronized, the formation and maintenance of a functional sperm reservoir improves the possibility of fertilization. In mammals, the oviduct regulates sperm functions, such as Ca2+ influx and processes associated with sperm maturation, collectively known as capacitation. A fraction of the stored sperm is released by unknown mechanisms and moves to the site of fertilization. There is an empirical association between the hormonal milieu in the oviduct and sperm detachment; therefore, we tested directly the ability of progesterone to induce sperm release from oviduct cell aggregates. Sperm were allowed to bind to oviduct cells or an immobilized oviduct glycan and then challenged with progesterone, which stimulated the release of 48% of sperm from oviduct cells or 68% of sperm from an immobilized oviduct glycan. The effect of progesterone on sperm release was specific; pregnenolone and 17α-OH-progesterone did not affect sperm release. Ca2+ influx into sperm is associated with capacitation and development of hyperactivated motility. Progesterone increased sperm intracellular Ca2+, which was abrogated by blocking the sperm-specific Ca2+ channel CatSper with NNC 055-0396. NNC 055-0396 also blocked the progesterone-induced sperm release from oviduct cells or immobilized glycan. An inhibitor of the non-genomic progesterone receptor that activates CatSper similarly blocked sperm release. This is the first report indicating that release of sperm from the sperm reservoir is induced by progesterone action through CatSper channels.

Redistribution of soluble N-ethylmaleimide-sensitive-factor attachment protein receptors in mouse sperm membranes prior to the acrosome reaction.

Formation of complexes between soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins on opposing membranes is the minimal requirement for intracellular membrane fusion. The SNARE, syntaxin 2, is found on the sperm plasma membrane and a second SNARE, vesicle associated membrane protein 2 (VAMP2, also known as synaptobrevin 2, SYB2), is on the apposing outer acrosomal membrane. During the acrosome reaction, the outer acrosomal membrane fuses at hundreds of points with the plasma membrane. We hypothesized that syntaxin 2 and VAMP2 redistribute within their respective membranes prior to the acrosome reaction to form trans-SNARE complexes and promote membrane fusion. Immunofluorescence and superresolution structured illumination microscopy were used to localize syntaxin 2 and VAMP2 in mouse sperm during capacitation. Initially, syntaxin 2 was found in puncta throughout the acrosomal region. At 60 and 120 min of capacitation, syntaxin 2 was localized in puncta primarily in the apical ridge. Although deletion of bicarbonate during incubation had no effect, syntaxin 2 puncta were relocated in the restricted region in less than 20% of sperm incubated without albumin. In contrast, VAMP2 was already found in puncta within the apical ridge prior to capacitation. The puncta containing syntaxin 2 and VAMP2 did not precisely co-localize at 0 or 60 min of capacitation time. In summary, syntaxin 2 shifted its location to the apical ridge on the plasma membrane during capacitation in an albumin-dependent manner but VAMP2 was already localized to the apical ridge. Puncta containing VAMP2 did not co-localize with those containing syntaxin 2 during capacitation; therefore, formation of trans-SNARE complexes containing these SNAREs does not occur until after capacitation, immediately prior to acrosomal exocytosis.