Fluorescent carbon nanodots (C-dots; 4.3 AE 0.8 nm) from fresh tender ginger juice provide high suppression of the growth of human hepatocellular carcinoma cells (HepG2), with low toxicity to normal mammary epithelial cells (MCF-10A) and normal liver cells (FL83B). The inhibition is selective to HepG2 over other tested cancer cells, including human lung cancer cell line (A549), human breast cancer cell line (MDA-MB-231), and human cervical cancer cell line (HeLa). Western blot results reveal that the C-dots up-regulate the expression of p53 protein only in the HepG2 cell line. The 50% inhibiting concentration (IC 50 ) value of the C-dots on HepG2 cells is 0.35 mg mL À1 . Image cytometry results show significant uptake of C-dots by HepG2 cells that induce intracellular production of reactive oxygen species (ROS, 18.2-fold increased), while other cells remain almost the same in ROS levels after treatment with C-dots (1.11 mg mL À1 ). The C-dots trigger the pro-apoptotic factor to promote HepG2 cell apoptosis. The C-dots effectively inhibit the growth of tumors in nude mice (104 AE 14 vs. 3.7 AE 0.2 mg with and without treatment within 14 days).

Summary The reproductive‐derived serine protease inhibitor Kazal‐type (Spink) has been identified in seminal plasma, and Spink–spermatozoa binding has been illustrated in many mammalian species including human. We used mice as experimental animal to study the mode of Spink action in the modulation of mammalian sperm activity. A Spink3‐binding zone was cytochemically stained on the sperm head at apical hook separated from intact acrosome, whether the cells were capacitated or not. The Spink3–spermatozoa binding neither changed the population of cells in the uncapacitated, capacitated and acrosome‐reacted status nor affected the capacitation‐related protein phosphorylation and cell motility enhancement. Despite that, the Spink–spermatozoa interaction resulted in decreasing the intracellular calcium concentration ([Ca2+]i) of the cell head and suppressing both the acrosome reaction induced by Ca+2 ionophore A23187 and the cell fertility. Furthermore, Spink3 seen on the head of spermatozoa in the uterine cavity after coitus could be removed by the trypsin‐like activity in the uterine fluid of oestrous females, and free Spink3 in the uterine cavity suppressed the protease activity. We integrated our data to shed light on the molecular mechanism of how Spink and its inhibiting protease are interplayed to modulate the activity of mammalian spermatozoa during their transit in the reproductive tract.

Formation of copulatory plugs by male animals is a common means of reducing competition with rival males. In mice, copulatory plugs are formed by the coagulation of seminal vesicle secretion (SVS), which is a very viscous and self-clotting fluid containing high concentration of proteins. In its native state, mouse SVS contains a variety of disulfide-linked high-molecular-weight complexes (HMWCs) composed of mouse SVS I-III, which are the major components of mouse SVS. Further, mouse SVS I-III are the substrates for transglutaminase 4 (TGM4), a cross-linking enzyme secreted from the anterior prostate. According to activity assays, mouse TGM4 prefers a mild reducing and alkaline environment. However, under these conditions, the activity of mouse TGM4 toward SVS I-III was much lower than that of a common tissue-type TGM, TGM2. On the other hand, mouse TGM4 exhibited much higher cross-linking activity than TGM2 when native HMWCs containing SVS I-III were used as substrates under non-reducing condition. By the action of TGM4, the clot of SVS became more resistant to proteolysis. This indicates that the activity of TGM4 can further rigidify the copulatory plug and extend its presence in the female reproductive tract. Together with the properties of TGM4 and the nature of its disulfide-linked SVS protein substrates, male mice can easily transform the semen into a rigid and durable copulatory plug, which is an important advantage in sperm competition.

We identified a testis-specific protease-like protein tentatively named TESPL and a pancreatic trypsinogen Prss2 from the clones of a yeast two-hybrid screen against a mouse testicular cDNA library using the trypsin inhibitor Spink3 from male accessory sexual glands as bait. The enzymatic motifs and the cysteine patterns in serine proteases are highly conserved in these two proteins. Based on the phylogenetic analysis, Prss2 duplicated recently and TESPL underwent distant evolution without gene duplication from the progenitor of trypsin-like and chymotrypsin-like proteases. We found that TESPL transcription was restricted to the testis and that the level of transcription was positively correlated with animal maturation. In contrast, Prss2 was constitutively expressed in many tissues including testis. Alignment of the cDNA-deduced sequences of serine proteases showed the replacement of an essential serine residue in the catalytic triad of serine proteases by a proline residue in TESPL, which was demonstrated to be a membrane-bound protein devoid of proteolytic activity. The immunohistochemical staining patterns of seminiferous tubules in the testis revealed TESPL mainly on postmeiotic cells such as spermatids and spermatozoa. On the mouse sperm from caudal epididymis, TESPL was localized mainly on the plasma membrane overlaying the acrosomal region. Further, orthology group for mouse TESPL was identified in the conserved gene family of eutherian testis serine protease 5.

Lysozyme (LYZ) c‐like proteins are primarily present in the testis and epididymis of male reproductive tissues. Here, we report a novel member of the c‐type LYZ family, the seminal vesicle‐secreted LYZ c‐like protein (SVLLP). Three forms of SVLLP were purified from mouse seminal vesicle secretions and characterized as glycoproteins with the same protein core but different N‐linked glycans. SVLLP is structurally similar to c‐type LYZ proteins. Only one of the 20 invariant residues was altered in the consensus sequence of c‐type LYZs; however, the changed residue (N53S) is one of two essential catalytic residues. LYZ activity assays demonstrated that the three glycoforms of SVLLP lacked enzyme activity. SVLLP is primarily expressed in seminal vesicles. Immunohistochemistry revealed that it occurs in the luminal fluid and mucosal epithelium of the seminal vesicles. Testosterone is not the primary regulator for its expression in the seminal vesicle. SVLLP binds to sperm and suppresses bovine serum albumin‐induced sperm capacitation, inhibits the acrosome reaction, and blocks sperm–oocyte interactions in vitro, suggesting that SVLLP is a sperm capacitation inhibitor.