[Introduction] Toll–like receptors (TLRs) are innate immune cell receptors which recognize pathogens during infection. Specifically, TLR2 pathway regulates calcium mobilization through the cell. Calcium influx is essential for acquisition of sperm fertilizing competence through regulation of capacitation and acrosome reaction (AR). Human sperm express TLR2 to recognize pathogens. Recently, we reported that TLR2 is localized in the posterior segment of bull sperm head. Here, we aimed to clarify the role of sperm TLR2 on sperm–oocytes interaction during in vitro fertilization (IVF). [Materials and Methods] Frozen–thawed bull sperm were washed and treated with TLR1/2 antagonist (0, 10 and 100 µM) for 30 min to block the sperm TLR2. Then, co–cultured with intact cumulus–oocyte complexes (COCs), denuded, or zona pellucida (ZP)–free oocytes for 6 h. After 1 h and 3 h of co–culture, the number of bound or penetrated sperm to the ZP were counted. Next, AR was induced using calcium ionophore–A23187 (CaA) to evaluate the effect of sperm TLR2 on calcium influx and AR by fluorescence microscopy and flow cytometry respectively. [Results] Co–culture of TLR2 antagonist-treated sperm either with COCs or denuded oocytes, but not with ZP–free oocytes, reduced the cleavage rate and blastocyst ratio. Computer–assisted sperm analysis (CASA) analysis revealed that motility parameters were not affected in TLR2 antagonist-treated sperm. However, TLR2 antagonist reduced the ability of sperm to bind or penetrate the ZP at 1 h and 3 h. Moreover, blockage of sperm TLR2 reduced the induction of AR either in the ZP–attached sperm or in CaA–triggered AR compared to control. Notably, exposure of TLR2 antagonist sperm to CaA clearly reduced the intracellular calcium level in sperm. Overall, the results provide evidence that sperm TLR2 is involved in sperm Ca²⁺ influx to induce AR which enables sperm to penetrate and fertilize oocytes in cows.

[Introduction] Modern techniques that require costly equipment such as microinjection and electroporation are generally used to produce the mutant porcine embryos. Our previous report showed that a lipofection-mediated gene-editing system without specialized equipment could be performed in zona pellucida (ZP)-free oocytes and embryos. However, the factors that affect the system efficiency need to be further examined. In the present study, we evaluated two factors affecting lipofection transfection efficiency involved in the timing of subject CRISPR/Cas9 system into ZP-free zygotes and the duration of lipofection treatment. [Materials and Methods] To examine adequate introduction timing, the zygotes collected at 5 h, 10 h, and 15 h post-in vitro fertilization (IVF) were incubated with lipofection reagent, guide RNA, and Cas9 for 5 h. Next, to optimize the exposure duration, the 10 h post-IVF-zygotes were incubated with lipofection reagent for 0 h, 2.5 h, 5 h, 10 h, and 20 h. The developmental capacity of treated embryos and the genotypes of the resulting blastocysts were evaluated. [Results] The lipofection treatment could be performed in 10 h, 15 h post-IVF zygotes for 5 h of incubation to generate the mutant blastocysts, and there were no detrimental effects on the developmental capacity among the treated zygotes. However, in the exposure duration treatment, the blastocysts formation rates and mutation rates of the resulting blastocysts decreased as the duration increased from 2.5 h to 20 h. In conclusion, a lipofection-mediated gene transfection system is feasible, particularly when the zygotes derived from 10 h post-IVF were treated with lipofection for 2.5 h to generate mutant blastocysts.

[Introduction] Expansion and folding in the neocortex are associated with unique cognitive abilities that distinguish human from other mammalian species. At present, a set of human- or primate-specific genes have been proven that promote cortical expansion and folding, such as TMEM14B. Since cortical folding emerges progressively during evolution, multiple genes, not only specific genes, but also conserved genes, should be involved in this process. In parallel, we have previously reported that promoter-associated non-coding RNAs (pancRNAs) transcribed from bidirectional promoter act on cis-acting elements in the transcriptional regulation of neighboring genes. Among such pancRNAs, we are focusing on pancCD63 because it is expressed in the human, but not in the mouse neural stem cells (NSCs). CD63, known as an exosomal marker, is also expressed much higher in human NSCs. [Materials and Methods] Human NSCs, AF22 cell line, were infected by lentivirus in knockdown experiments. Immunostaining detecting active caspase3 and cell cycle labeling assay using EdU were performed to analyze cell apoptosis and proliferation, respectively. As a gain-of-function experiment, in-utero electroporation was performed on E13.5 mouse embryos, and plasmid DNA was microinjected through the uterus into the lateral ventricle. [Results] Knockdown of pancCD63 reduced expression level of CD63, suggesting that pancCD63 can be as a regulatory molecule to affect CD63 expression. Knockdown of either CD63 or pancCD63 resulted in a dramatical decrease in EdU+ cells and an increase in active caspase3+ cells, which suggests that pancCD63-CD63 pair promote human NSC proliferation. Overexpression of CD63 in mouse brain increased number of basal progenitors marked by Pax6. It is to be noted that, at E18.5, CD63 overexpression generated a large number of upper layer neuron and showed a folding-like structure. In summary, pancCD63-CD63 pair plays a key role in cortical development and folding.

[Introduction] Kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) of the hypothalamus are considered to regulate gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) surge in female rodents. Our previous studies showed that purinergic signaling involves GnRH/LH surge and then ovulation in female rats. The present study, thus, aims to identify the purinergic neurons that project to the vicinity of AVPV kisspeptin neurons.[Materials and method] Adult Wistar-Imamichi female rats (n = 4) were stereotaxically injected with fluorogold (FG), a retrograde tracer, into the AVPV. Then, the animals were ovariectomized and primed with diestrous levels of estradiol-17β (E2) for 5 days followed by proestrous levels of E2 for 2 days. Seven days after the FG injection, the animals were perfused with 4% paraformaldehyde and 50-μm brain sections including the hypothalamus and hindbrain were made using a cryostat. The brain sections were subjected to double-immunohistochemistry for FG and vesicular nucleotide transporter (VNUT), a marker of purinergic neurons. [Results] VNUT-immunoreactive cells were mainly found in the hypothalamic arcuate nucleus and medial part of supramammillary nucleus (SuM), and brainstem A1, A2, A6 regions and area postrema (AP). FG- and VNUT-co-immunoreactive cells were found in the majority of VNUT-ir cells in the A1, around one-third of VNUT-ir cells in the A2, one-tenth of VNUT-ir cells in the A6, and few VNUT-ir cells in the SuM. These results suggest that the purinergic neurons located in the A1, and A2 may project their axons to the vicinity of AVPV kisspeptin neurons and possibly play a role in an induction of GnRH/LH surge, and then ovulation in female rats.

[Introduction] CCL2 (C-C motif chemokine ligand 2), which is known to recruit immune cells to the sites of inflammation, has been reported to be expressed in the bovine endometrium. We have reported that CCL2 induced proliferation and stimulated the expression of prostaglandin (PG) synthases in cultured endometrial cells. However, the details of how CCL2 involved in the implantation mechanism are still unclear. The purpose of present study is to analyze the functional properties of CCL2 in the bovine endometrium and embryo. [Materials and Methods] Bovine endometrial tissue, epithelial (BEE) and stromal (BES) cells, and bovine blastocyst-derived trophoblastic (BT) cells were used for the analysis. Recombinant bovine CCL2 was used for in vitro experiments. Gene expressions were analyzed by RT-PCR or qPCR. [Results] Since the previous results showed that both expressions of PGESs and PGFSs were high, we focused on the analysis of PG transporter genes (MRP4 and PGT). The amount of MRP4 and PGT were significantly high in CCL2 treated BEE and BES in vitro. The mRNA of chemokine receptors (CCR1, CCR2 and CXCR3) were detected in BT. The amount of PCNA and IFNt were significantly high in BT treated with CCL2. CCL2 significantly increased the attachment rate of BT vesicles to BEE in in vitro co-culture system. The amount of OPN increased in BEE, and ICAM-1 increased both in BEE and BT by CCL2 treatment. These results indicated that CCL2 has the potential to regulate the circulation of PGs in bovine endometrium and embryo growth. In addition, CCL2 has a possibility of regulating the process of bovine embryo attachment to endometrium by modulating expression of binding molecules.