Rumex crispus is a perennial plant that grows in humid environments across Korea. Its roots are used in traditional Korean medicine to treat several diseases, including diseases of the spleen and skin and several inflammatory pathologies. In this study, different solvent fractions (n-hexane, dichloromethane, ethyl acetate, n-butanol, and aqueous fractions) from an ethanol extract of R. crispus roots were evaluated for the presence and composition of anthraquinone compounds and antioxidants by checking for such things as free radical scavenging activity, and electron and proton atom donating ability. In addition, anti-inflammatory activity was measured by NO scavenging activity and inflammatory cytokine production; furthermore, anti-cancer activity was measured by apoptosis-inducing ability. Polyphenolic and flavonoid compounds were shown to be abundant in the dichloromethane and ethyl acetate fractions, which also exhibited strong antioxidant activity, including free radical scavenging and positive results in FRAP, TEAC, and ORAC assays. HPLC analysis revealed that the dichloromethane fractions had higher anthraquinone contents than the other fractions; the major anthraquinone compounds included chrysophanol, emodin, and physcione. In addition, results of the anti-inflammatory assays showed that the ethyl acetate fraction showed appreciable reductions in the levels of nitric oxide and inflammatory cytokines (TNF-α, IL-1β, and IL-6) in Raw 264.7 cells. Furthermore, the anthraquinone-rich dichloromethane fraction displayed the highest anticancer activity when evaluated in a human hepatoma cancer cell line (HepG2), in which it induced increased apoptosis mediated by p53 and caspase activation.

The glycosylphosphatidylinositol‐anchored sperm hyaluronidases (Hyals), sperm adhesion molecule 1 (SPAM1) and HYAL5, have long been believed to assist in sperm penetration through the cumulus‐oocyte complex (COC), but their role in mammalian fertilization remains unclear. Previously, we have shown that mouse sperm devoid of either Spam1 or Hyal5 are still capable of penetrating the COC and that the loss of either Spam1 or Hyal5 alone does not cause male infertility in mice. In the present study, we found that Spam1/Hyal5 double knockout (dKO) mice produced significantly fewer offspring compared with wild‐type (WT) mice, and this was due to defective COC dispersal. A comparative analysis between WT and Spam1/Hyal5 dKO epididymal sperm revealed that the absence of these 2 sperm Hyals resulted in a marked accumulation of sperm on the outside of the COC. This impaired sperm activity is likely due to the deficiency in the sperm Hyals, even though other somatic Hyals are expressed normally in the dKO mice. The fertilization ability of the Spam1/Hyal5 dKO sperm was restored by adding purified human sperm Hyal to the in vitro fertilization medium. Our results suggest that Hyal deficiency in sperm may be a significant risk factor for male sterility.—Park, S., Kim, Y.‐H., Jeong, P.‐S., Park, C., Lee, J.‐W., Kim, J.‐S., Wee, G., Song, B.‐S., Park, B.‐J., Kim, S.‐H., Sim, B.‐W., Kim, S.‐U., Triggs‐Raine, B., Baba, T., Lee, S.‐R., Kim, E. SPAM1/HYAL5 double deficiency in male mice leads to severe male subfertility caused by a cumulus‐oocyte complex penetration defect. FASEB J. 33, 14440‐14449 (2019). www.fasebj.org

Glycosylphosphatidylinositol-anchored sperm hyaluronidases have long been believed to assist in sperm penetration through the cumulus–oocyte complex (COC); however, their role in mammalian fertilization remains unclear. Previously, we have shown that hyaluronidase 5 (Hyal5)/Hyal7double-knockout (dKO) mice produce significantly fewer offspring than their wild-type (WT) counterparts because of defective COC dispersal. Male infertility is mainly caused by a low sperm count. It can be further exacerbated by the deficiency of sperm hyaluronidase, which disperses the cumulus cells of the outer layer of the COC. In the current study, we evaluated the effects of a low count ofHyal-deficient sperm and conditions of ovulated oocytes on the fertilization rate using a mouse model. Our results demonstrated that a low sperm count further decreases thein vitrofertilization (IVF) rate ofHyal-deficient dKO spermatozoa. In addition, the dKO spermatozoa resulted in a fertilization rate of 12.5% upon fertilizing COCs with a thick cumulus layer, whereas the IVF rate was comparable to that of WT spermatozoa when oocytes with a thin or no cumulus layer were fertilized. Finally, we proved that the IVF rate of dKO spermatozoa could be recovered by adding rat spermatozoa as a source of sperm hyal. Our results suggest that a deficiency of proteins involved in fertilization, such as sperm hyal, has a vital role in fertilization.

To elucidate the functional role of V-set and immunoglobulin domain-containing 1 (VSIG1) in spermatogenesis and fertilization, we knocked out (KO) VSIG1 in a mouse embryo using CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) -mediated genome editing. Reverse transcription PCR was performed using cDNA synthesized from VSIG1 KO testis RNA. Although Western blot analysis using a specific antibody to VSIG1 confirmed VSIG1 protein defects in the KO mice, hematoxylin-eosin staining analysis was similar in the KO and wild-type mice. Additionally, computer-assisted sperm analysis and in vitro fertilization experiments were conducted to confirm the activity and fertilization ability of sperm derived from the KO mouse. Mice lacking VSIG1 were viable and had no serious developmental defects. As they got older, the KO mice showed slightly higher weight loss, male mice lacking VSIG1 had functional testes, including normal sperm number and motility, and both male and female mice lacking VSIG1 were fertile. Our results from VSIG1 KO mice suggest that VSIG1 may not play essential roles in spermatogenesis and normal testis development, function, and maintenance. VSIG1 in sperm is dispensable for spermatogenesis and male fertility in mice. As several genes are known to possess slightly different functions depending on the species, the importance and molecular mechanism of VSIG1 in tissues of other species needs further investigation.