An in vitro study of the effect of mifepristone and ulipristal acetate on human sperm functions.

Ulipristal acetate (UPA) and mifepristone are currently well-established agents for emergency contraception. Both drugs are selective progestogen receptor modulators which have been shown to have better efficacy than the widely used levonorgestrel in prevention of pregnancy. However, there is only limited information on the action of UPA on sperm function. The present study compared the in vitro biological effects of mifepristone and UPA on human sperm functions. Spermatozoa from semen samples with normal semen parameters were isolated. Capacitated spermatozoa were pre-incubated with 0.04, 0.4, 4 and 40 µM mifepristone or UPA for 1 h. Sperm motility, viability, DNA integrity, capacitation, spontaneous acrosome reaction, spontaneous hyperactivation, zona pellucida (ZP) binding capability and intracellular calcium concentration ([Ca(2+)]i) were determined. The effects of mifepristone and UPA on progesterone-induced acrosome reaction, hyperactivation and [Ca(2+)]i were also studied. Our results showed that mifepristone and UPA dose-dependently suppressed progesterone-induced acrosome reaction, hyperactivation and [Ca(2+)]i at concentrations ≥0.4 µM in human spermatozoa. Both compounds did not affect sperm motility, viability, DNA integrity, capacitation, spontaneous acrosome reaction, spontaneous hyperactivation, ZP binding capability and [Ca(2+)]i. This study demonstrated that UPA and mifepristone modulate human sperm functions by acting as progesterone antagonists. The results enable us to gain a better understanding of the mechanisms by which mifepristone and UPA work for emergency contraception, and provide a scientific basis for their clinical application.

Glycodelin-A modulates syncytialization of human BeWo choriocarcinoma cell line.

Cytotrophoblasts are the key trophoblast cells which differentiate into different trophoblast lineages. In this report, glycodelin-A action on fusion of a cytotrophoblast-like cell line (BeWo) was investigated. It significantly reduced the spontaneous fusion of BeWo cells. The treatment enhanced the invasion and extracellular-signal regulated kinases activation of BeWo cells. The mRNA expression of syncytialization markers, human chorionic gonadotrophin and glial cells missing homolog 1 were suppressed upon glycodelin-A treatment. The data suggest a possible function of glycodelin-A in mediating cytotrophoblast differentiation.

Effect of leptin on motility, capacitation and acrosome reaction of human spermatozoa.

Leptin is a polypeptide hormone with important roles in reproduction. It has been detected in human seminal plasma as well as on human ejaculated spermatozoa. This study aimed at studying the possible role of leptin in regulating human sperm functions. Immunofluorescent staining was used to study the expression of leptin and its receptor. The correlation between the concentration of leptin and soluble leptin receptor (ObRs) in seminal plasma as measured by enzyme-linked immunosorbant assay and sperm motility parameters measured by computer-assisted sperm analysis (CASA) was determined. The effects of recombinant leptin on human sperm motility, capacitation and acrosome reaction as measured by chlortetracycline staining were also studied. Leptin immunoreactivity was demonstrated at the equatorial and neck regions of human spermatozoa, whereas that of ObRs was shown up on the tail. After Percoll separation, spermatozoa with high density had more intense leptin immunoreactivity compared with those with low density. No significant correlation was found between seminal plasma concentration of leptin/ObRs and sperm motility parameters. After incubation with recombinant human leptin for either 3 h or overnight, there was no change in all the CASA motility parameters determined and percentages of capacitated and acrosome-reacted spermatozoa. We concluded that leptin does not have a significant effect on motility and capacitation/acrosome reaction in human ejaculated mature spermatozoa. Its role in male reproduction is yet to be determined.

Glycosylation related actions of glycodelin: gamete, cumulus cell, immune cell and clinical associations.

Glycodelin is an example of a glycoprotein whose complex-type glycans mediate biological actions in human reproduction and immune reactions. Being attached to an identical protein backbone, glycodelin oligosaccharides vary significantly from one reproductive tissue to another and have an effect on its own secretion and role in cell communication. For instance, uterine glycodelin-A inhibits sperm-oocyte interaction by binding on the sperm head. This is a glycosylation-dependent phenomenon, in which fucosyltransferase-5 plays a key role. Glycodelin-S from seminal plasma binds evenly around the sperm head and maintains an uncapacitated state in the spermatozoa, until the isoform is detached during sperm passage through the cervix. Glycodelin-F from follicular fluid and Fallopian tube binds to the acrosomal region of the sperm head, thereby inhibiting both the sperm-oocyte binding and premature progesterone-induced acrosome reaction. The cumulus cells surrounding the oocyte can capture glycodelin-A and -F from the surrounding environment and convert these isoforms to a cumulus cell isoform, glycodelin-C. It differs by glycosylation from the other isoforms, and it too attaches on the sperm head, with the highest density in the equatorial region. Glycodelin-C is capable of detaching the sperm-bound inhibitory isoforms so that the sperm-oocyte binding is enhanced. Glycodelin-A also has immunosuppressive actions directed to cellular, humoral and innate immunity. Although these actions depend mainly on the protein backbone, glycosylation also plays a part. Glycosylated glycodelin may be involved in the protection of spermatozoa against maternal immune reactions, and glycodelin also has apoptogenic activity. Some glycosylation patterns of glycodelin may mask its apoptogenic domain. This review updates the recent research and clinical associations of glycodelin, highlighting the role of glycosylation.

The contribution of D-mannose, L-fucose, N-acetylglucosamine, and selectin residues on the binding of glycodelin isoforms to human spermatozoa.

Previous data showed that glycodelin-A from amniotic fluid and glycodelin-F from follicular fluid inhibited sperm-zona pellucida binding. Solubilized zona pellucida reduced the binding of glycodelin-F to sperm extract dose dependently. This study demonstrated that the zona pellucida proteins also reduced the binding of glycodelin-A to sperm extract. Ionophore-induced acrosome reaction reduced the binding of iodinated glycodelin-A and -F to sperm, indicating that the glycodelin-binding sites are on the outer acrosomal membrane or on the sperm plasma membrane overlying the acrosome. While the binding of glycodelin-A to sperm was suppressed by mannose and fucose neoglycoproteins, that of glycodelin-F was also reduced by acetylglucosamine neoglycoprotein. Pretreatment of sperm with inhibitors of mannosidase and acetylglucosaminidase reduced the binding of glycodelin-F to sperm. On the other hand, inhibitor of mannosidase but not of acetylglucosaminidase inhibited the binding of glycodelin-A. In a competition binding assay, mannosidase reduced both glycodelin-A and -F binding whereas acetylglucosaminidase reduced only glycodelin-F binding. While fucosidase reduced the binding of both glycodelins, fucosidase inhibitor was marginally active in suppressing the binding of glycodelins to human sperm. Among the selectins tested, only E-selectin had a slight inhibitory effect on the binding of glycodelin-A to sperm. The binding of glycodelin-F was unaffected by selectins and their antibodies. In conclusion, the binding of glycodelin-A to sperm involves mannose, fucose, and possibly E- selectin residues, while that of glycodelin-F involves mannose, fucose, and N-acetylglucosamine but not the selectin residue.

Zona-binding inhibitory factor-1 from human follicular fluid is an isoform of glycodelin.

Zona-binding inhibitory factor-1 (ZIF-1), a glycoprotein in human follicular fluid, reduces the binding of spermatozoa to the zona pellucida. ZIF-1 has a number of properties similar to those of glycodelin-A from human follicular fluid. The objective of this study was to compare the biochemical characteristics of these two glycoproteins. N-terminal sequencing and protease-digested peptide mapping showed that ZIF-1 and glycodelin-A have the same protein core. However, these glycoproteins differ in their oligosaccharide chains, as demonstrated by fluorophore-assisted carbohydrate electrophoresis, lectin-binding ability, and isoelectric focusing. ZIF-1 inhibited spermatozoa-zona pellucida binding slightly more than did glycodelin-A and significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. Indirect immunofluorescence staining revealed specific binding of glycodelin-A and ZIF-1 to the acrosome region of human spermatozoa, where ZIF-1 produced a stronger signal than did glycodelin-A at the same protein concentration. These data suggest that ZIF-1 is a differentially glycosylated isoform of glycodelin that potently inhibits human sperm-egg interaction. Future study on the function role of ZIF-1 would provide a better understanding of the regulation of fertilization in humans.

Comparative study of the biological activity of spermatozoa-zona pellucida binding inhibitory factors from human follicular fluid on various sperm function parameters.

Previous studies showed that human follicular fluid (hFF) from gonadotrophin stimulated cycles contained two glycoproteins, named as ZIF-1 and ZIF-2, that reduced the zona binding capacity of spermatozoa. The present study showed that the spermatozoa-zona pellucida binding inhibitory activity was also present in hFF from natural cycle. Using the hemizona binding assay, the inhibitory effect of ZIF-1 on the zona binding capacity of spermatozoa was dose-dependent. The effect of ZIF-2 was also dose-dependent, in the range of 10-100 ng/ml. The inhibitory effects of both ZIF-1 and -2 increased with the duration of the spermatozoa-ZIF interaction. The effect of the former was present up to 120 min incubation, whilst that of latter occurred for the first 90 min. The zona binding inhibitory effect of ZIF-1 and -2 was additive when they were used together to treat the spermatozoa. The biological activity of ZIFs on other sperm parameters that might affect spermatozoa-zona pellucida binding was also investigated. ZIF-1 did not affect the acrosomal status of human spermatozoa while ZIF-2 significantly increased the number of acrosome reacted spermatozoa in the range of 0.1-10 microg. However, the increase in the incidence of acrosome-reacted spermatozoa after ZIF-2 treatment could not totally account the inhibitory effect of ZIF-2 on zona binding. Both glycoproteins did not affect the motility of human spermatozoa. Radioactively-labelled ZIFs bound to human spermatozoa. Unlabelled ZIF displaced the bound radioactivity of spermatozoa treated with the corresponding labelled ZIF. These suggested the presence of specific binding sites of ZIFs on human spermatozoa.

The synthesis and fate of glycodelin in human ovary during folliculogenesis.

The ontogeny of glycodelin in human ovarian follicles during folliculogenesis was studied. Glycodelin immunoreactivity began to be detected in the granulosa cells and thecal cells of late secondary follicles. Immunoreactivity was also found in both the luteinized granulosa cells and cumulus cells obtained from women undergoing the assisted reproduction treatment. However, only the luteinized granulosa cells, and not the cumulus cells, expressed glycodelin mRNA. Results also showed that the cumulus cells took up radiolabelled glycodelin and partially deglycosylated some of it. Glycodelin (and a partially deglycolsylated form of glycoldelin) appeared to complex with two cytoplasmic or membrane components of the cumulus cells. The data also demonstrated that ZIF-1, a glycoprotein isolated from human follicular fluid, was immunologically similar to glycodelin. In conclusion, we suggest that glycodelin is synthesized in the granulosa cells of ovarian follicles at late secondary follicle stage. It then may be released into the follicular fluid from where it is taken up and partially modified by the cumulus cells.