Woojin Kang

Functional Roles of Mouse Sperm Hyaluronidases, HYAL5 and SPAM1, in Fertilization

Although sperm entry into the oocyte-cumulus complex and subsequent sperm penetration through the cumulus matrix to reach the oocyte zona pellucida are essential for mammalian fertilization, the molecular mechanism remains controversial. Previously, we have shown that mouse sperm lacking SPAM1 are capable of penetrating the cumulus matrix despite a delayed dispersal of cumulus cells. We also have identified another sperm hyaluronidase, HYAL5, as a candidate enzyme involved in sperm penetration through the cumulus. In the present study, we produced HYAL5-deficient mice to uncover the functional roles of HYAL5 and SPAM1 in fertilization. The HYAL5-deficient mice were fully fertile and yielded normal litter sizes. In vitro fertilization assays demonstrated that HYAL5-deficient epididymal sperm is functionally normal. We thus conclude that HYAL5 may be dispensable for fertilization. Comparative analysis among wild-type, HYAL5-deficient, and SPAM1-deficient epididymal sperm revealed that only SPAM1 is probably involved in sperm penetration through the cumulus matrix. Notably, the loss of SPAM1 resulted in a remarkably increased accumulation of sperm on the surface or outer edge of the cumulus. These data suggest that SPAM1 may function in sperm entry into the cumulus and sperm penetration through the cumulus matrix.

Mice Lacking Two Sperm Serine Proteases, ACR and PRSS21, Are Subfertile, but the Mutant Sperm Are Infertile In Vitro

Although sperm serine protease and proteasome have long been believed to play an important role in the fertilization process, the molecular mechanism is still controversial. In this study, we have produced double-knockout mice lacking two sperm serine proteases, ACR and PRSS21, to uncover the functional role of the trypsinlike activity in fertilization. The double-knockout male mice were subfertile, likely owing to the incompleteness of fertilization in the oviductal ampulla. Despite male subfertility, the mutant epididymal sperm exhibited the inability to undergo acrosomal exocytosis on the zona pellucida (ZP) surface and to traverse the ZP, thus resulting in the failure of fertilization in vitro. The double-knockout epididymal sperm were also defective in penetration through the cumulus matrix to reach the ZP. When epididymal sperm were artificially injected into the uterus of wild-type mice, the 2-cell embryos, which had previously been fertilized by double-knockout sperm, were recovered at a low but significant level. The mutant epididymal sperm were also capable of fertilizing the oocytes in the presence of uterine fluids in vitro. These data demonstrate that the trypsinlike protease activity of ACR and PRSS21 is essential for the process of sperm penetration through the cumulus matrix and ZP in vitro, and suggest that the female reproductive tract partially compensates for the loss of the sperm function. We therefore conclude that the sperm trypsinlike activity is still important but not essential for fertilization in vivo in the mouse.

Biogenesis of sperm acrosome is regulated by pre-mRNA alternative splicing of Acrbp in the mouse

Significance Mammalian sperm possess a Golgi-derived exocytotic organelle, the acrosome, located on the apical region of the head. Proper biogenesis of the acrosome is essential for the fertilization process because the aberrant acrosome formation results in the sterility or subfertility of males. Here, we show that the acrosome formation is governed by two forms of proacrosin-binding protein ACRBP, wild-type ACRBP-W and variant ACRBP-V5, which are generated by pre-mRNA alternative splicing of Acrbp. ACRBP-V5 is involved in the formation and configuration of the acrosomal granule during early spermiogenesis, whereas the inactive status of proacrosin in the acrosome is maintained by ACRBP-W until acrosomal exocytosis. Proper biogenesis of a sperm-specific organelle, the acrosome, is essential for gamete interaction. An acrosomal matrix protein, ACRBP, is known as a proacrosin-binding protein. In mice, two forms of ACRBP, wild-type ACRBP-W and variant ACRBP-V5, are generated by pre-mRNA alternative splicing of Acrbp. Here, we demonstrate the functional roles of these two ACRBP proteins. ACRBP-null male mice lacking both proteins showed a severely reduced fertility, because of malformation of the acrosome. Notably, ACRBP-null spermatids failed to form a large acrosomal granule, leading to the fragmented structure of the acrosome. The acrosome malformation was rescued by transgenic expression of ACRBP-V5 in ACRBP-null spermatids. Moreover, exogenously expressed ACRBP-W blocked autoactivation of proacrosin in the acrosome. Thus, ACRBP-V5 functions in the formation and configuration of the acrosomal granule during early spermiogenesis. The major function of ACRBP-W is to retain the inactive status of proacrosin in the acrosome until acrosomal exocytosis.

Ubiquitin-activating enzyme E1 inhibitor PYR-41 retards sperm enlargement after fusion to the egg

The ubiquitin-proteasome system, which is initiated by a single ubiquitin-activating enzyme E1 (UBE1), is involved in male reproduction via spermatogenesis and function in mammals. Here we explored the influence of UBE1-specific inhibitor, 4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester (pyrazone-41 or PYR-41) in female reproduction. UBE-1 was detected by immunoblotting and immunocytochemistry in mouse eggs and was localized mainly under the egg plasma membrane. PYR-41 pretreatment suppresses the development of eggs into two-cell embryos. Specifically, pretreatment retarded sperm enlargement and meiotic chromosomal division after sperm-egg fusion. PYR-41 pretreatment disturbed β-catenin, a well-known target protein for ubiquitination, localization under the egg plasma membrane and on spindle microtubules in wild-type eggs. Otherwise, PYR-41 treatment had no effect on the two-cell development of eggs lacking β-catenin. Our results raise the possibility that inhibition of the ubiquitin-proteasome system suppresses sperm enlargement through impaired β-catenin-mediated mechanism.

Seminal vesicle proteins SVS3 and SVS4 facilitate SVS2 effect on sperm capacitation.

Mammalian spermatozoa acquire their fertilizing ability in the female reproductive tract (sperm capacitation). On the other hand, seminal vesicle secretion, which is a major component of seminal plasma, inhibits the initiation of sperm capacitation (capacitation inhibition) and reduces the fertility of the capacitated spermatozoa (decapacitation). There are seven major proteins involved in murine seminal vesicle secretion (SVS1-7), and we have previously shown that SVS2 acts as both a capacitation inhibitor and a decapacitation factor, and is indispensable for in vivo fertilization. However, the effects of SVSs other than SVS2 on the sperm have not been elucidated. Since mouse Svs2-Svs6 genes evolved by gene duplication belong to the same gene family, it is possible that SVSs other than SVS2 also have some effects on sperm capacitation. In this study, we examined the effects of SVS3 and SVS4 on sperm capacitation. Our results showed that both SVS3 and SVS4 are able to bind to spermatozoa, but SVS3 alone showed no effects on sperm capacitation. On the other hand, SVS4 acted as a capacitation inhibitor, although it did not show decapacitation abilities. Interestingly, SVS3 showed an affinity for SVS2 and it facilitated the effects of SVS2. Interaction of SVS2 and spermatozoa is mediated by the ganglioside GM1 in the sperm membrane; however, both SVS3 and SVS4 had weaker affinities for GM1 than SVS2. Therefore, we suggest that separate processes may cause capacitation inhibition and decapacitation, and SVS3 and SVS4 act on sperm capacitation cooperatively with SVS2.

Degradation of phosphate polymer polyP enhances lactic fermentation in mice

In bacteria, a polymer of inorganic phosphate (Pi) (inorganic polyphosphate; polyP) is enzymatically produced and consumed as an alternative phosphate donor for adenosine triphosphate (ATP) production to protect against nutrient starvation. In vertebrates, polyP has been dismissed as a “molecular fossil” due to the lack of any known physiological function. Here, we have explored its possible role by producing transgenic (TG) mice widely expressing Saccharomyces cerevisiae exopolyphosphatase 1 (ScPPX1), which catalyzes hydrolytic polyP degradation. TG mice were produced and displayed reduced mitochondrial respiration in muscles. In female TG mice, the blood concentration of lactic acid was enhanced, whereas ATP storage in liver and brain tissues was reduced significantly. Thus, we suggested that the elongation of polyP reduces the intracellular Pi concentration, suppresses anaerobic lactic acid production, and sustains mitochondrial respiration. Our results provide an insight into the physiological role of polyP in mammals, particularly in females.

Autophagy-disrupted LC3 abundance leads to death of supporting cells of human oocytes

Autophagic recycling of cell parts is generally termed as the opposite of cell death. Here, we explored the relation between cell death and autophagy by examining granulosa cell layers that control oocyte quality, which is important for the success of fertilization. Granulosa cell layers were collected from infertile women and morphologically divided into four types, viz., mature (MCCs), immature (ICCs), and dysmature cumulus cells (DCCs), and mural granulosa cells (MGCs). Microtubule-associated protein light chain 3 (LC3), which is involved in autophagosome formation, was expressed excessively in DCCs and MGCs, and their chromosomal DNA was highly fragmented. However, autophagy initiation was limited to MGCs, as indicated by the expression of membrane-bound LC3-II and autophagy-related protein 7 (ATG7), an enzyme that converts LC3-I to LC3-II. Although pro-LC3 was accumulated, autophagy was disabled in DCCs, resulting in cell death. Our results suggest the possibility that autophagy-independent accumulation of pro-LC3 proteins leads to the death of human granulosa cells surrounding the oocytes and presumably reduces oocyte quality and female fertility.

Birthweights and Down syndrome in neonates that were delivered after frozen-thawed embryo transfer: The 2007-2012 Japan Society of Obstetrics and Gynecology National Registry data in Japan

To evaluate the use of frozen embryos on the outcome of assisted reproductive technology (ART), a retrospective study of the Japanese Assisted Reproductive Technology Registry data during the years 2007‐2012 was conducted.