Yuan-qing Yao

Effects of human follicular fluid on the capacitation and motility of human spermatozoa

OBJECTIVE To investigate the capacitation and motility kinetics of spermatozoa treated with human follicular fluid (FF). DESIGN Controlled, experimental laboratory study. SETTING University-based gynecology unit. PATIENT(S) Human FF was collected from women undergoing assisted reproductive treatment. Semen samples were obtained from men visiting subfertility clinics. INTERVENTION(S) Spermatozoa were incubated with human FF under various experimental conditions. Spermatozoa incubated with Earles balanced salt solution were used as the control. MAIN OUTCOME MEASURE(S) Chlortetracycline staining patterns and sperm motility parameters. RESULT(S) The rate of capacitation in the human FF-treated spermatozoa was significantly higher than that in the control spermatozoa after 1 hour and 3 hours of treatment. The percentage of acrosome-reacted spermatozoa also was significantly higher after human FF treatment than after control treatment. These effects of human FF were dose-dependent. Human FF-treated spermatozoa maintained their velocities at the zero-hour level for 5 hours, whereas the velocities of the control spermatozoa decreased significantly after 1 hour. Human FF treatment significantly increased the beat cross-frequency above the rate at zero hour for 5 hours. The hyperactivation of the human FF-treated spermatozoa remained stable for 3 hours, whereas that of the control spermatozoa decreased significantly after 1 hour of incubation. Significantly more human FF-treated spermatozoa underwent hyperactivation than did control spermatozoa after 1 hour and 3 hours of treatment. The effects of human FF on beat cross-frequency and hyperactivation were dose-dependent. CONCLUSION(S) Human FF promotes capacitation and the acrosome reaction within a short period. It also stimulates or maintains various sperm motility parameters.

Human oviductal cells produce a factor(s) that maintains the motility of human spermatozoa in vitro

OBJECTIVE To characterize in part the factor(s) in conditioned medium (CM) that maintains sperm motility after human oviductal cell culture. DESIGN Controlled, experimental, laboratory study. SETTING University-based gynecology unit. PATIENT(S) Fallopian tubes were obtained from patients who underwent tubal ligation or hysterectomy. Semen with normal sperm parameters was obtained from men who visited subfertility clinics. INTERVENTION(S) Spermatozoa were incubated with CM and their motility was evaluated by a computer-aided sperm analysis system. MAIN OUTCOME MEASURE(S) Curvilinear velocity, straight-line velocity, average path velocity, linearity, amplitude of lateral head displacement, beat cross-frequency, and percentage of spermatozoa that exhibited hyperactivation. RESULT(S) Compared with their baseline motility (0 hour), spermatozoa incubated with CM maintained various motility parameters for a longer period than did control spermatozoa. All the motility parameters of the CM-treated spermatozoa were higher than those of the control spermatozoa at the same time point. This effect of CM was dose-dependent and increased with the duration of incubation. The effect was stable at 56 degrees C but was not observed after 100 degrees C heat treatment. Trypsin, but not proteinase K, abolished the effect. A fraction with a molecular weight of <3 kd in the CM was responsible for the observed effect. CONCLUSION(S) Human oviductal cells produce a peptide(s) that maintains sperm motility.

Effects of human oviductal cell coculture on various functional parameters of human spermatozoa

OBJECTIVE To investigate the effect of human oviductal cells on various sperm functions in vitro. DESIGN Controlled experimental laboratory study. SETTING University gynecology unit. PATIENT(S) Women undergoing tubal ligation or hysterectomy and men who were visiting our subfertility clinics. INTERVENTION(S) Coculture of oviductal cells with human spermatozoa in vitro; sperm functions were determined after coculture. MAIN OUTCOME MEASURE(S) Capacitation, acrosome reaction, zona binding, and oocyte fusion. RESULT(S) Oviductal cells and conditioned medium induced more spermatozoa to capacitate than did control medium. Although there was no difference in the spontaneous acrosome reaction between any of the groups, the coculture group had a lower percentage of acrosome-reacted spermatozoa after calcium ionophore challenge than did the control and conditioned medium groups. Coculture and conditioned medium treatment reduced the number of spermatozoa bound to the zona pellucida. The penetration rate and penetration index of the control spermatozoa in the zona-free hamster oocyte penetration test were significantly higher than that of the cocultured or conditioned medium-treated spermatozoa. CONCLUSION(S) Human oviductal cells promoted capacitation, stabilized the acrosome, and suppressed binding to the zona pellucida and fusion with the oocyte in vitro.

Identification of mRNAs that are up-regulated after fertilization in the murine zygote by suppression subtractive hybridization.

Transcriptions occur in mouse preimplantation embryos as early as one-cell stage. However, our understanding on gene expression at this stage is lacking. The present study applied suppression subtractive hybridization (SSH) to compared gene expression profiles of mouse zygote and oocyte. Forty-four differentially expressed genes were selected and shown positive signals by reverse dot-blot hybridization. DNA sequences comparison of these putative clones with the GenBank/EMBL databases using BLAST search identified 38 clones with >90% identity to known genes and six novel clones with less than 70% homology to the databases. Eleven out of the 44 differentially expressed clones were either originally isolated from male embryo or testis-specific genes, suggesting that these genes may be derived from paternal genome. Five differentially expressed genes of interest, including bromodomain-containing protein BP75, spindlin, radixin, pituitary tumor-transforming gene (PTTG), and proteoglycan core protein (serglycin) were further studied by semi-quantitative RT-PCR. It is noted that spindlin which involves in cell division is highly expressed in zygote, suggesting that this protein may play an important role in zygotic gene activation (ZGA) and early stage development in 1-cell stage mouse embryos.

Profiles of sperm morphology and motility after discontinuous multiple‐step Percoll density gradient centrifugation

Summary. Morphology and motility are important parameters for assessing the fertilizing capacity of spermatozoa. This investigation reports a systematic study on the profiles of these parameters after Percoll gradient centrifugation. Spermatozoa from normal human semen were fractionated by discontinuous Percoll gradients (30%, 45%, 75%, 90%). Spermatozoa washed with Earles balanced salt solution were used as a control. After centrifugation, sperm morphology was evaluated according to strict criteria; motility was assessed by a computer‐assisted semen analysis system. The results showed that the percentage of spermatozoa with normal morphology increased, while those with severe morphological defects decreased, as the density of Percoll increased. The percentages of motile spermatozoa and hyperactivated spermatozoa, and the velocity and amplitude of lateral head displacement of the spermatozoa were significantly higher in the 75% and 90% Percoll fractions than that in the 30% and 45% ones, and in the control. These results demonstrated that Percoll density gradient centrifugation enriched spermatozoa in terms of morphology and motility.

Soluble Human Leukocyte Antigen-G5 Activates Extracellular Signal-Regulated Protein Kinase Signaling and Stimulates Trophoblast Invasion

Soluble human leukocyte antigen-G (HLA-G) is a non-classical class Ib HLA molecule that is secreted from blastocysts. Soluble HLA-G modulates the immune tolerance of the mother and can be used as a prognostic factor for the clinical pregnancy rate. However, the underlying mechanism of how soluble HLA-G5 affects pregnancy remains largely unknown. We hypothesized that soluble HLA-G5 promotes successful implantation and pregnancy by modulating trophoblast invasion through receptor binding and activation of extracellular signal-regulated protein kinase (ERK) signaling pathway. Recombinant HLA-G5 protein over-expressed in E. coli BL21 was purified to near homogeneity. We studied the expression of HLA-G5 and its receptors, the leukocyte immunoglobulin-like receptor subfamily B1 (LILRB1) and killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), in primary trophoblasts and trophoblastic (JAr and JEG-3) cell lines by florescence-labeled HLA-G5. HLA-G5 was detected in the primary trophoblasts and JEG-3 cells. The LILRB1 and KIR2DL4 receptors were expressed in both primary trophoblasts and trophoblastic cell lines. HLA-G5 stimulated cell invasion (p<0.05) and increased urokinase (uPA) and matrix metalloproteinases (MMPs) transcripts and their activity (p<0.05) in trophoblastic cells. HLA-G5 activated the ERK signaling pathway and induced ERK1/2 phosphorylation in the trophoblastic cell lines. Addition of ERK inhibitors (U0126 and PD98059) nullified the stimulatory effect of HLA-G5 on trophoblastic cell invasion. Taken together, HLA-G5 induced trophoblast invasion by binding to KIR2DL4 and LILRB1, by increasing uPA and MMPs expressions and by activating the ERK signaling pathway.

Effects of human follicular fluid on spermatozoa that have been cocultured with human oviductal cells

OBJECTIVE To investigate the sequential effects of human oviductal cells and human follicular fluid (hFF) on various sperm functions. DESIGN Laboratory experimental study. SETTING University gynecology unit. PATIENT(S) Fallopian tubes were from patients undergoing tubal ligation or hysterectomy. Semen was from men attending the subfertility clinics. INTERVENTION(S) Spermatozoa were treated with [1] 6 hours in Earles balanced salt solution (EBSS-BSA; control); [2] 5 hours in EBSS-BSA and 1 hour with hFF (hFF); [3] 5 hours with oviductal cells and 1 hour in EBSS-BSA (coculture); and [4] 5 hours with oviductal cells and 1 hour with hFF (sequential). MAIN OUTCOME MEASURE(S) Motility, acrosome reaction, zona binding, and oocyte fusion. RESULT(S) Groups II and III spermatozoa had similar motility and were better than that of group I. Group IV displayed higher motility parameters than the other groups. Human follicular fluid induced acrosome reaction. The incidence of acrosome reaction in group IV was significantly lower than that in group II. Group III did not affect the acrosome reaction. Spermatozoa in groups II-IV had lower zona binding capacity than those in group I. Human follicular fluid stimulated oocyte penetration, whereas oviductal cells suppressed this effect of hFF. CONCLUSION(S) Oviductal cells maintained the fertilizing capacity of spermatozoa, whereas hFF facilitated the fertilization process of oviductal spermatozoa.