No abstract

The participation of acrosin in mammalian sperm penetration through the zona pellucida has been amply debated. In this paper we report the immunolocalization--by silver enhanced immunogold technique using ACRO-8C10 monoclonal antibody to human acrosin--of proacrosin/acrosin on ejaculated rabbit spermatozoa incubated in vitro in a capacitating medium and on spermatozoa recovered from the perivitelline space. After incubation in a capacitating medium, four different patterns were observed: (1) no labeling on acrosome intact spermatozoa; (2) labeling on the rim of the head; (3) labeling on the whole acrosome area; and (4) no labeling on acrosome reacted spermatozoa. At the start of incubation, spermatozoa with pattern 1 were the most abundant, whereas at the end of the 32 h incubation period, patterns 2 and 3 were the most frequent. On the other hand, 625 perivitelline spermatozoa were recovered from 17 fertilized rabbit eggs, of which 26% were labeled with the antiacrosin monoclonal antibody ACRO-8C10 in two different areas: (1) only on the equatorial region; and (2) only on the postacrosomal area. These results are consistent with the idea that proacrosin/acrosin remains associated to the acrosome reacted spermatozoa for long periods of time, and that proacrosin/acrosin associated to perivitelline spermatozoa could be responsible for the second penetration of fresh rabbit eggs by perivitelline spermatozoa.

No abstract

ABSTRACT. The present work identifies and quantifies the morphological alterations of scallop Argopecten purpuratus spermatozoa caused by long-term cryopreservation. Percentages of motility, fertilization and injured spermatozoa were quantified by optic microscopy and scanned electron microscopy. These parameters were evaluated in sperm without treatment (CTR), spermatozoa incubated in cryoprotective solution but not freezed (ICS) and freezed-thawed spermatozoa (FTS). Spermatozoa of ICS treatment remained motile longer than those of CTR, whereas those of FTS treatment were lowest. Morphology of the spermatozoa was affected in several ways by the freeze-thawing treatment; some had their head deformed or swollen, others had their cell membrane folded or broken; acrosome reaction; anomalous positions or absence of mitochondria as well as broken, stiff or loss of lineal structure of tail. CTR and ICS treatments had higher percentages of undamaged sperm (87.7% and 79.0% respectively), while FTS samples had 14.2% of undamaged sperm. The tail was the spermatic structure most commonly injured in FTS (77.0%), the percentage of sperm with head injury was 55.1% and with acrosome reaction was 28.7%, whereas middle piece was affected in 23.9% of sperm. Percentages of fertilization were 68.3%, 67.9% and 58.2% for CTR, ICS and FTS respectively, which were not significantly different. There was a higher correlation between injuries and motility than between injuries and fertilization success. Correlation between motility and fertilization was low (0.605 and 0.668 with motility at 5 and 30 min, respectively). Keywords: cryopreservation, Argopecten purpuratus, scallop, sperm, Chile. Alteraciones morfológicas en espermatozoides criopreservados de concha de abanicoArgopecten purpuratus RESUMEN. El presente trabajo identifica y cuantifica las alteraciones morfológicas en espermatozoides de concha de abanico A. purpuratus causadas por la criopreservación en nitrógeno líquido. Porcentajes de motilidad, fecundación de ovocitos frescos y espermatozoides lesionados (en cabeza, acrosoma, pieza media y flagelo) fueron determinados bajo microscopía óptica y electrónica de barrido. Estos parámetros fueron evaluados en un control sin tratamiento (CTR), espermatozoides incubados en solución crioprotectora pero sin congelamiento (ICS) y espermatozoides congelados-descongelados (FTS). Los espermatozoides del tratamiento ICS mostraron mayor motilidad que de CTR, mientras que la motilidad de los espermatozoides del tratamiento FTS fue la más baja. La morfología externa de los espermatozoides fue afectada de varias formas por el congelamiento-descongelamiento; cabeza deformada o hinchada, membrana celular plegada o rota, reacción acrosómica, anormal posición o ausencia de las mitocondrias, ruptura, rigidez o pérdida de la estructura lineal del flagelo. El CTR e ICS presentó los mayores porcentajes de espermatozoides ilesos (87,7% y 79,0% respectivamente), mientras que las muestras de FTS tuvieron 14,2% de espermatozoides ilesos. El flagelo fue la e...

The efficiency of producing embryos using in vitro technologies in livestock species rarely exceeds the 30–40% threshold, indicating that the proportion of oocytes that fail to develop after in vitro fertilization and culture is considerably large. Considering that the intrinsic quality of the oocyte is one of the main factors affecting blastocyst yield, the precise identification of noninvasive cellular or molecular markers that predict oocyte competence is of major interest to research and practical applications. The aim of this review was to explore the current literature on different noninvasive markers associated with oocyte quality in the bovine model. Apart from some controversial findings, the presence of cycle-related structures in ovaries, a follicle size between 6 and 10 mm, large number of surrounding cumulus cells, slightly expanded investment without dark areas, large oocyte diameter (>120 microns), dark cytoplasm, and the presence of a round and smooth first polar body have been associated with better competence. In addition, the combination of oocyte and zygote selection via brilliant cresyl blue (BCB) test, spindle imaging, and the anti-Stokes Raman scattering microscopy together with studies decoding molecular cues in oocyte maturation have the potential to further optimize the identification of oocytes with better developmental competence for in-vitro-derived technologies in livestock species.

No abstract

Acrosin, an acrosomal serine protease, has been associated with binding of spermatozoa and their penetration through the zona pellucida. This study was aimed at determining whether the remaining proacrosin/acrosin system on rabbit perivitelline spermatozoa still has proteolytic activity and whether this activity is involved in further penetration of unfertilised rabbit eggs. Eight hundred and sixty-five rabbit perivitelline spermatozoa were evaluated by the gelatin-substrate film technique for the detection of acrosin on individual spermatozoan. Fifteen per cent of the studied spermatozoa showed small digestion halos on the gelatin film. The proteolytic activity of rabbit perivitelline spermatozoa was inhibited in the presence of 1 mg/ml of soybean trypsin inhibitor (SBTI) or with 20 μg/ml of a mixture of the monoclonal anti-proacrosin/acrosin antibody. In vitro fertilisation occurred in 21.8% of rabbit oocytes co-incubated with perivitelline spermatozoa and was completely inhibited when oocytes were incubated with 600 μg/ml of a mixture of three anti-acrosin monoclonal antibodies (ACRO-A8C10, ACRO-C2B10 and ACRO-C5F10). Inseminations in the presence of anti-cholera monoclonal antibody (irrelevant to spermatozoa) resulted in 17.6% fertilisation. These results support the idea that the residual proacrosin/acrosin system in perivitelline spermatozoa might be involved in spermatozoal binding and/or second penetration through the zona pellucida.

The aim of this study was to evaluate the effect of Lepidium meyenii (maca) in chemically and physically subfertile mice. After 35 days, the following groups of mice were evaluated: control, sham, chemical subfertility, chemical subfertility-maca-supplemented, physical subfertility, physical subfertility-maca-supplemented and maca-supplemented only. Motility (32.36% ± 5.34%) and sperm count (44.4 ± 5.37 × 10(6) /ml) in the chemically and physically subfertile mice (11.81% ± 4.06%, 17.34 ± 13.07 × 10(6) /ml) decreased compared to the control (75.53% ± 2.97% and 57.4 ± 19.6 10(6) /ml) and sham (53.5% ± 7.86% and 58.4 ± 14.10 10(6) /ml). Maca was able to reverse the deleterious effect of motility (76.36 ± 1.97) as well as sperm count (53.5 ± 9.18 × 10(6) /ml) on chemical subfertility. In contrast, maca did not reverse the effects of induced physical subfertility nor motility (18.78% ± 14.41%) or sperm count (20.17 ± 11.20 × 10(6) /ml). The percentage of sperm DNA fragmentation in the physically subfertile mice increased (11.1% ± 19.29%) compared to the control (0.84% ± 0.85%). However, in the physically subfertile group, maca decreased sperm DNA fragmentation (2.29% ± 2.30%) closer to the sham (1.04% ± 0.62%) and the control (0.84% ± 0.85%). The group supplemented only with maca showed 0.54% ± 0.50% of spermatozoa with DNA fragmentation. Yet, the differences observed were statistically not significant. In conclusion, it appears that maca activates the cytochrome P450 system after chemically induced subfertility. However, it does not reverse the low mitochondrial membrane potential in spermatozoa compromised in the physical subfertility group.