The aim of this study was to investigate effects of hyaluronidase during IVM on oocyte maturation, oxidative stress status, expression of cumulus expansion-related (PTX, pentraxin; GJA1, gap junction protein alpha 1; PTGS2, prostaglandin-endoperoxide synthase 2) and fatty acid metabolism-related (FADS1, delta-6 desaturase; FADS2, delta-5 desaturase; PPARα, peroxisome proliferator-activated receptor-alpha) mRNA, and embryonic development of porcine oocytes. The cumulus-oocyte complexes (COCs) were incubated with 0.1 mg/mL hyaluronidase for 44 h. Cumulus expansion was measured at 22 h after maturation. At 44 h after maturation, nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured. Gene expression in cumulus cells was analyzed using real time PCR. The cleavage rate and blastocyst formation were evaluated at Day 2 and 7 after insemination. In results, expansion of cumulus cells was suppressed by treatment of hyaluronidase at 22 h after maturation. Intracellular GSH level was reduced by hyaluronidase treatment (p < 0.05). On the other hand, hyaluronidase increased ROS levels in oocytes (p < 0.05). Only PTGS2 mRNA was enhanced in COCs by hyaluronidase (p < 0.05). Population of oocytes reached at metaphase II stage was higher in control group than hyaluronidase treated group (p < 0.05). Both of cleavage rate and blastocyst formation were higher in control group than hyaluronidase group (p < 0.05). Our present results showed that developmental competence of porcine oocytes could be reduce by hyaluronidase via inducing oxidative stress during maturation process and it might be associated with prostaglandin synthesis. Therefore, we suggest that suppression of cumulus expansion of COCs could induce oxidative stress and decrease nuclear maturation via reduction of GSH synthesis and it caused to decrease developmental competence of mammalian oocytes.

When sperm penetrates into the ovum, hyaluronidase plays a role of hydrolyzing the hyaluronic acid present in the membrane surrounding the oocytes. The zona pelucida of the ovum is hydrolysed to facilitate sperm entry. Therefore, the aim of this study was to investigate the effects of hyaluronidase during the in vitro maturation in porcine oocytes. The cumulus-oocyte complexes (COCs) were cultured during in vitro maturation (IVM) medium containing 0 and 0.1mg/ml hyaluronidase for 44 h. Representative images of oocytes were captured after cultured for 0 h and 22 h by using a microscope. The area was quantified using a image J software. After 44 h of IVM, nuclear maturation stage was assessed by the aceto-orcein method. In results, cumulus cells expansion was no significant difference between control and hyaluronidase treatment groups in 0 h. However, after 22 h of IVM, in 0.1mg/ml hyaluronidase group, cumulus cells diffusion was significantly reduced than control group (p<0.05). After 22 h matured COCs, the cumulus cells were normally expanded in the control group, but there was a significantly lower 0.1mg/ml hyaluronidase group than control group (p<0.05). The nuclear maturation rate was treated with 0.1mg/ml hyaluronidase, it was significantly decrease than control group (p<0.05). In conclusion, our study indicated that hyaluronidase exposure could reduce nuclear maturation in vitro by reducing the expansion of cumulus cells. According to the results, we conjectured that hyaluronidase treatment disrupted the oocyte maturation by hydrolyzing the hyaluronic acid around the oocytes and it reduces the activity of the intercellular gap junction because it weakens cumulus cell bonds and interferes with communication. However, additional studies on hyaluronidase are needed. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Education) (2016R1D1A1B03931746).

Sperm adhesion molecule 1 (SPAM1) and Hyaluronidase 5 (HYAL5) has been well-known as assistants for sperm penetrate through the cumulus mass surrounding the ovulated eggs. However, so far their role in mammalian fertilization remain elusive, because mouse sperm lacking SPAM1 or HYAL5 were still capable of penetrating the cumulus mass despite a delayed dispersal of cumulus mass. Those data collectively demonstrated that SPAM1 or HYAL5 deficiency alone was not sufficient to cause male infertility in mice. In the present study, SPAM1 and HYAL5-simultaneous deficient male mice model was generated. Because of inhibition in sperm hyaluronidases, SPAM1 and HYAL5-deficient male mice produced significantly smaller numbers of offspring than hetero type and wild type mice. Hyaluronic acid degradation assay and cumulus oocyte complex dispersal assay as well as sperm motility assay using double knock out sperm and extracts had severe adverse effects on the dispersal of cumulus oocyte complex, which was the main reason for the impaired fertility of double knock out male sperm. Moreover, hyaluronic acid degradation assay using human sperm extracts revealed that sperm hyaluronidase has a principal role in sperm penetration through the cumulus oocyte complex. In conclusion, our results suggest that sperm hyaluronidase deficiency may be sufficient to cause male sterility in mammal because SPAM1 and HYAL5 deficiency sperm not impaired the sperm motility in hyaluronic acid but also cumulus oocyte complex penetration.

During mammalian fertilization, germ cell-specific hyaluronidases, such as sperm adhesion molecule 1 (SPAM1) and hyaluronoglucosaminidase 5 (Hyal5), are important for the dispersal of the cumulus mass. In this study, we demonstrated that bull Hyal5 is a single copy gene on chromosome 4 that is expressed specifically in the testis. In addition, we expressed recombinant bull SPAM1 and Hyal5 in human embryonic kidney 293T cells and showed that these enzymes possessed hyaluronidase activity. We also demonstrated that a polyclonal antibody against bull sperm hyaluronidase inhibits sperm-egg interactions in an in vitro fertilization (IVF) assay. Our results suggested that bull Hyal5 may have a critical role in bull fertilization.

In this study, the anti-inflammatory effect of hyaluronidase (HAase) inhibition was determined from 92 species of oriental herbal medicine extracted with water and ethanol solvents because of their non-toxicity in the human body. The water extracts of Evodia officinalis (86.8%), Thuja orientalis (80.8%), Carthami semen (66.5%), Melia azedarach (74.7%), Siegesbeckia pubescens (61.3%), Saururus chinensis (49.15%) showed a relatively greater anti-inflammatory activity. The ethanol extracts of Ailanthus altissima and Saururus chinensis demonstrated the highest anti-inflammatory effect at above 90%. Saururus chinensis was selected for its high anti-inflammatory effect in both water and ethanol extract. Ethanol was more effective than water and optimal extraction conditions for phenolic compounds was determined to be extraction with 50% ethanol for 12 hours. The extracts from Saururus chinensis in optimal condition showed 70~80% anti-inflammatory effect when 100~250 μg/mL phenolic concentration was treated. Concentration of above 500 μg/mL decreased the inhibitory effect. The anti-inflammatory effect and extraction yield were increased by ultra-fine grind technology, indicating that this method can be used to increase the extraction yield of phenolic compounds from medicinal plants.