The effect of betaine for mouse sperm cryopreservation.

Sperm cryopreservation is an effective method of preserving male fertility in humans, as well as domestic and experimental animals. However, various factors such as ice crystal formation, osmotic stress, and oxidative stress, negatively influence the motility and viability of post-thawed spermatozoa. Betaine, which works as an osmoprotectant is known to work as a nontoxic cryoprotectant. However, the protective effects during mouse sperm cryopreservation are still unclear. Thus, the purpose of this study was to investigate whether betaine has protective effects during the process of mouse sperm cryopreservation. In this study, betaine was found to be effective in maintaining sperm motility during the freezing procedure and 1% (85.4 mM) betaine was identified as the optimal concentration to be added to cryopreservation solutions. It was also found that betaine improves the integrity of the plasma membranes of sperm tails, suggesting that betaine has a positive effect on sperm motility.

Nuclear-to-cytoplasmic ratios of 1PN and 2PN zygotes after in vitro fertilization of mouse oocytes.

Numerous studies have reported comparisons of the nuclear-to-cytoplasmic (NC) ratio during mitosis. However, little information is known about how the pronuclear size is regulated and determined at the end of meiosis II in mammalian zygotes. The present study aims to analyze the NC ratio of female and male pronuclei, and also to compare the size of single pronuclei using photographs that were obtained during experiments to create chimeric hermaphrodites from 2-cell oocytes. The volume of both the female and the male pronucleus was found to correlate with the volume of the oocyte cytoplasm. The NC ratio of the male pronucleus was greater than that of the female pronucleus. The NC ratio of the average volume of the female and male pronuclei was greater than the NC ratio of the mononucleate oocytes. The occurrence of 1PN oocytes was significantly higher when the volume of cytoplasm was lower than the cut-off value. These results indicated that the NC ratio is retained during pronuclear formation. A higher NC ratio in male compared with the female pronucleus indicated structural and/or molecular difference between the two pronuclei. 1PN formation may occur when sperm enters close to the MII spindle.

Effect of docosahexaenoic acid on in vitro growth of bovine oocytes.

PURPOSE: The present study investigated the effects of docosahexaenoic acid (DHA) on the growth of bovine oocytes. METHODS: Oocytes and granulosa cell complexes (OGCs) were collected from early antral follicles (0.4-0.7 mm) on the surface of ovaries harvested from a slaughterhouse. The OGCs were cultured with 0, 1, and 10 µmol/L docosahexanoic acid (DHA) for 16 days. RESULTS: Antrum formation of the OGCs and the number of granulosa cells (GCs) surrounding the oocytes were comparable among groups, whereas supplementation of 0.1 µmol/L of DHA significantly improved oocyte growth. Oocytes grown with DHA had a higher fertilization rate, acetylation levels of H4K12, and ATP contents, as well as a lower lipid content compared with those grown without DHA. In addition, GCs surrounding OGCs grown with DHA had low lipid content compared with vehicle counterparts. Furthermore, when GCs were cultured in vitro, DHA increased ATP production, mitochondrial membrane potential, and reduced lipid content and levels of reactive oxygen species. RNA-seq of GCs revealed that DHA increased CPT1A expression levels and affect genes associated with focal adhesion, oxidative phosphorylation, and PI3K-AKT etc. CONCLUSION: The results suggest that DHA supplementation affects granulosa cell characteristics and supports oocyte growth in vitro.

Upregulation of CHOP participates in caspase activation and virus release in human astrovirus-infected cells.

Human astroviruses (HAstVs), non-enveloped RNA viruses with positive-sense RNA genomes, are an important cause of acute gastroenteritis in young children, although the processes that produce infectious virions are not clearly defined. To track the viral replication complex (RC) upon HAstV1 infection, the subcellular distribution of double-stranded (ds) RNA and of ORF1b, a viral RNA polymerase, was examined by immunocytochemistry. Foci that were positive for dsRNA and for ORF1b were co-localized, and both foci were also co-localized with resident proteins of the endoplasmic reticulum (ER). Focusing on the association between the HAstV RC and ER, we examined the expression of unfolded protein response (UPR) markers and found that targets of eukaryotic translation initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4), including CCAAT/enhancer-binding protein homologous protein (CHOP), a proapoptotic transcription factor, were upregulated at the late phase in HAstV-infected cells. Consistently, eIF2α was phosphorylated at the late phase of HAstV infection. The formation of foci resembling stress granules, another known downstream response to eIF2α phosphorylation, was also observed at the same period. Phosphorylation of eIF2α was attenuated in protein kinase R (PKR)-knockdown cells, suggesting that, unlike the canonical ER stress response, PKR was involved in eIF2α phosphorylation in response to HAstV infection. Studies have indicated that immature HAstV capsid protein is processed by caspases, and caspase cleavage is integral to particle release. Inhibition of CHOP upregulation reduced caspase activation and the release of HAstV RNA from cells during HAstV infection. Our results suggest that the eIF2α-ATF4-CHOP pathway participates in HAstV propagation.

Relationship between the number of cells surrounding oocytes and energy states of oocytes.

Lipid content, ATP content, and histone acetylation are thought to reflect the energy state of cells. In addition, the energy state closely associates with growth and developmental ability of oocytes. Oocyte growth is accompanied by active proliferation of the surrounding granulosa cells (GCs), and GCs play a key role in the provision of energy substrates to the oocytes. In the present study, we first examined the relationship among the average number of GCs per follicle, the average number of cumulus cells (CCs) per oocyte, and the average lipid content in oocytes that developed in vivo within individual donor gilts. Second, we validated the relationship between the number of cells surrounding oocytes and the energy states of oocytes by using an IVC system of oocyte granulosa cell complexes (OGCs) derived from early antral follicles. We collected cumulus cells and oocyte complexes (COCs) from antral follicles (3-5 mm in diameter) and found that average lipid content in oocytes significantly correlated with the average number of both GCs/follicle and CCs/oocyte (P < 0.05). In the next series of experiments, we collected OGCs from early antral follicles (0.5-0.7 mm in diameter), and cultured them for 14 days, and then determined the cell number of OGCs, as well as the lipid content, ATP content, and acetylation level of H4K12 in oocytes grown in vitro. In addition, glucose consumption by OGCs was calculated from the sample media collected at Days 13 and 14. The lipid content of oocytes grown in vitro, significantly correlated with the number of cells surrounding the oocytes (P < 0.01) and with the level of glucose consumption by OGCs (P < 0.01). In addition, both ATP content and H4K12 acetylation levels of oocytes grown in vitro significantly correlated with the number of cells surrounding the oocytes (P < 0.05) and glucose consumption by OGCs (P < 0.05). In conclusion, the lipid content of oocytes depends on the number of cells surrounding the oocytes, and glucose uptake by OGCs is crucial for lipid content and ATP content, and H4K12 acetylation in oocytes.

Gene expression patterns in granulosa cells and oocytes at various stages of follicle development as well as in in vitro grown oocyte-and-granulosa cell complexes.

Follicle development is accompanied by proliferation of granulosa cells and increasing oocyte size. To obtain high-quality oocytes in vitro, it is important to understand the processes that occur in oocytes and granulosa cells during follicle development and the differences between in vivo and in vitro follicle development. In the present study, oocytes and granulosa cells were collected from early antral follicles (EAFs, 0.5-0.7 mm in diameter), small antral follicles (SAFs, 1-3 mm in diameter), large antral follicles (LAFs, 3-7 mm in diameter), and in vitro grown oocyte-and-granulosa cell complexes (OGCs), which were cultured for 14 days after collection from EAFs. Gene expression was analyzed comprehensively using the next-generation sequencing technology. We found top upstream regulators during the in vivo follicle development and compared them with those in in vitro developed OGCs. The comparison revealed that HIF1 is among the top regulators during both in vivo and in vitro development of OGCs. In addition, we found that HIF1-mediated upregulation of glycolysis in granulosa cells is important for the growth of OGCs, but the cellular metabolism differs between in vitro and in vivo grown OGCs. Furthermore, on the basis of comparison of upstream regulators between in vivo and in vitro development of OGCs, we believe that low expression levels of FLT1 (VEGFA receptor), SPP1, and PCSK6 can be considered causal factors of the suboptimal development under in vitro culture conditions.

Examination of a plasmid-based reverse genetics system for human astrovirus.

A plasmid-based reverse genetics system for human astrovirus type 1 (HAstV1) is examined. Upon transfection into 293T cells, the plasmid vector, which harbors a HAstV1 expression cassette, expressed astroviral RNA that appeared to be capable of viral RNA replication, as indicated by the production of subgenomic RNA and capsid protein expression irrespective of the heterologous 5' ends of the transcribed RNA. Particles infectious to Caco-2 cells were made in this system; however, their infectivity was much lower than would be expected from the amount of particles apparently produced. Using Huh-7 cells as the transfection host with the aim of improving viral capsid processing for virion maturation partially restored the efficiency of infectious particle formation. Our results support the possibility that the DNA transfection process induces a cellular response that targets late, but not early, stages of HAstV1 infection.

Effects of reaggregated granulosa cells and oocytes derived from early antral follicles on the properties of oocytes grown in vitro.

In this study, we examined the effects of reconstructed oocyte-granulosa cell complexes (OGCs) on the development of porcine oocytes derived from early antral follicles (EAFs; 0.5-0.7 mm in diameter). When denuded oocytes were cocultured with granulosa cells derived from other EAFs, the oocytes and granulosa cells aggregated to form OGCs after 2 days of culture. After 14 days of culture, we compared cell number, oocyte diameter, and oocyte chromatin configuration in unmanipulated (natural) OGCs, reconstructed OGCs, and OGCs collected from antral follicles (AFs, 3.0-6.0 mm in diameter). The diameters of oocytes from reconstructed OGCs grown in vitro were not different from those of oocytes from natural OGCs, although they were significantly smaller than those of oocytes from antral follicle (AF) OGCs. Oocyte chromatin configuration did not differ among the 3 OGC groups, but the oocyte nuclear maturation rate was lower in the reconstructed OGCs and higher in the AF OGCs. However, when the in vitro culture period for the reconstructed OGCs was extended by 2 days, the nuclear maturation rate of oocytes from reconstructed OGCs was similar to that of oocytes from natural OGCs. In addition, blastocysts were successfully obtained from oocytes from reconstructed OGCs. In conclusion, we established an innovative culture method that allows oocytes and granulosa cells from EAFs to reaggregate as reconstructed OGCs, which yield oocytes with the ability to develop to the blastocyst stage.

Relationship between mitochondrial DNA copy number and SIRT1 expression in porcine oocytes.

The present study assessed the effect of resveratrol on the expression of SIRT1 and mitochondrial quality and quantity in porcine oocytes. Supplementing the maturation medium with 20 µM resveratrol increased the expression of SIRT1, and enhanced mitochondrial functions, as observed from the increased ATP content and mitochondrial membrane potential. Addition of resveratrol also improved the ability of oocytes to develop into the blastocyst stage following activation. The effects of resveratrol on mitochondrial number were examined by comparing the mitochondrial DNA copy number (Mt number) between group of oocytes collected from the same donor gilt ovaries. Supplementing the maturation medium with only resveratrol did not affect the Mt number in the oocytes. However, supplementing the maturation medium with 10 µM MG132, a proteasome inhibitor, significantly increased the amount of ubiquitinated proteins and Mt number by 12 and 14%, respectively. In addition, when resveratrol was added to the medium containing MG132, the Mt number increased significantly by 39%, this effect was diminished by the addition of the SIRT1 inhibitor EX527. Furthermore, supplementing the medium with MG132 and EX527 did not affect Mt number. The mean SIRT1 expression in 20 oocytes was significantly and positively correlated with the Mt number in oocytes collected from the same donor. This study suggests that the expression of SIRT1 is associated with the Mt number in oocytes. In addition, activation of SIRT1 by resveratrol enhances the biosynthesis and degradation of mitochondria in oocytes, thereby replenishing and improving mitochondrial function and the developmental ability of oocytes.

Estradiol has a major role in antrum formation of porcine preantral follicles cultured in vitro.

Antrum formation and estradiol (E2) secretion occur during early folliculogenesis. The objective was to determine the role of E2 in antrum formation of oocyte-granulosa cell complexes (OGCs) derived from porcine preantral follicles (PAFs). Supplementation of the culture medium with E2 (1 µg/mL) improved antrum formation of OGCs during 14 days of in vitro culture. Furthermore, adding 0.1 µg/mL androstenedione (a precursor of E2) to the medium also improved antrum formation. Concentration of E2 was higher in the medium of developmentally competent OGCs versus incompetent OGCs (8.5 vs. 3.5 ng/mL, P < 0.05). Fulvestrant (1 µg/mL), a competitive inhibitor of E2, completely inhibited antrum formation of OGCs that were cultured in medium containing either E2 (0.1 µg/mL) or androstenedione (0.1 µg/mL); however, increasing E2 to 1 µg/mL ameliorated the inhibitory effect. Conversely, in the case of early antral follicles, OGCs formed antrums without E2 supplementation. After E2 pretreatment, OGCs derived from PAFs formed antrums even when the OGCs were subsequently cultured in medium without E2. Furthermore, when OGCs derived from PAFs were cultured without E2 followed by an additional in vitro culture with E2, antrums were formed, albeit with the same period delay by the same pretreatment periods. In conclusion, E2 in the culture medium was indispensable for in vitro antrum formation of OGCs derived from PAFs; therefore, one of the roles of E2 is in the initiation of antrum formation.