Heterologous Fertilization Research Articles

The Prion‐like Protein Doppel Enhances Ovine Spermatozoa Fertilizing Ability

The function of prion-like protein Doppel was suggested to be related to male fertility. In this study, the importance of ovine Doppel polypeptide on spermatozoa capacitation and fertilization was evaluated. After refolding, recombinant Doppel (rDpl) was supplemented with different concentrations (40, 80 or 190 ng/ml) to ovine spermatozoa during the capacitation process. In experiment 1, post-thawed ovine spermatozoa were incubated with different concentrations of rDpl during 1 h for swim-up, and changes in sperm motility, concentration, vigour, viability and capacitation were monitored (10 replicates). In experiment 2, the fertilization ability of post-swim-up spermatozoa incubated as above was tested through heterologous fertilization of bovine in vitro matured oocytes (n = 423, three replicates). Regardless of dosage, rDpl improved (p ≤ 0.03) spermatozoa viability. Sperm individual motility and vigour were the highest (p ≤ 0.04) for the group receiving 190 ng/ml rDpl. Sperm supplemented with the highest doses of rDpl achieved higher (p = 0.02) fertilization rates (56.0 ± 3.0%) than control (39.1 ± 2.2%) and 40 ng/ml rDpl (39.8 ± 2.7%). Preliminary data suggest that Doppel protein may enhance in vitro spermatozoa fertilizing ability.

Protamine Withdrawal from Human Sperm Nuclei Following Heterologous ICSI into Hamster Oocytes

During late stages of spermatogenesis in mammals, most histones bound to DNA are replaced by protamines (PRM), which results in formation of supercondensed and genetically inert sperm chromatin. At fertilization, mature spermatozoon penetrates oocyte and chromatin is remodeled "back" from nucleoprotamine to nucleohistone state. While being crucial for activation of male genome and ultimately for initiation of embryonic development, this process is poorly studied, especially in humans. Data on model animals concerning PRM to histones exchange post fertilization are few and contradictory. As direct experimentation with human embryos is impossible due to ethical, legal and technical reasons, we evaluate the timing and mode of PRM removal in a heterologous ICSI system using hamster ova injected with human sperm. Localization of human PRM 1 and 2 in hybrid zygotes was established using immunofluorescence. We observed a marked zygote to zygote variability in male pronuclei size for any time point post ICSI and demonstrated that PRM removal correlates with the developing pronuclei area rather than time after injection. Overall, the disappearance of protamines from sperm is rather rapid and most likely completed within 1 hr. We propose that the critical characteristic influencing PRM removal after heterologous fertilization is the intrinsic heterogeneity of the human sperm population. The same yet unexplored variance may be one of the reasons for canceled, delayed or aberrant early embryonic development during natural or artificial fertilization in humans.

In vitro and in vivo fertility of ram semen cryopreserved in different extenders

Seminal traits of frozen-thawed (FT) ram semen and in vitro and field fertility in native Portuguese breeds were evaluated in 4 experiments. In exp. 1 and 2 the cryopreservation capacity of 2 extenders, E1 (15% egg yolk-EY) and E2 (4.5% EY and trehalose) was compared through morphological evaluation and in vitro fertilizability of FT ram semen. Exp. 3 aimed to determine the usefulness of in vitro homologous/heterologous fertilization tests as tools for predicting ram fertility. Exp. 4 was conducted to verify if the identified differences between the 2 extenders could be confirmed by field fertility. E1 showed a better cryoprotective action expressed by higher in vitro and field fertility results. In conclusion, EY is difficult to be replaced in ram semen extenders. Heterologous fertilization seems to be a useful tool for predicting fertility of FT ram semen.

Assisted Procreation and its Relationship to Genetics and Eugenics

The article below is intended to reflect on whether or not a eugenic tendency constitutes an intrinsic element of human fertilization in vitro. The author outlines ideas and circumstances which characterized the foundation and propagation of eugenics between the eighteenth and nineteenth centuries. A brief discussion follows on some of the standard procedures of in vitro fertilization, and in particular, those which manifest a trace or hint of eugenics--heterologous fertilization and sperm banking, preimplantation genetic diagnosis (PGD) and embryo selection--practices which, nonetheless, are used on a large scale and shed light on both the essence of procreative medicine and on the current cultural environment. The objective of the article is to explore whether it is possible to eliminate the eugenic connotations without foregoing the benefits of technical and scientific progress.

Development of Heat-Shocked Tilapia Eggs Following Activation by Water Immersion or Heterologous Fertilization: Implications for the Initiation of Gynogenesis

An attempt was made in this study to initiate artificial gynogenesis in tilapia eggs by water activation, as an alternative to heterologous fertilization. Blue tilapia Oreochromis aureus (Steindachner) eggs activated by water immersion and heat-shocked at 42 oC failed to develop into gynogens. However, heat shock of eggs fertilized with normal or irradiated carp sperm developed into putative gynogens. Un-shocked normal or irradiated carp-fertilized eggs died shortly after hatching due to haploid condition or hybrid inviability. Results of this study indicate that, sperm contact is necessary for successful initiation of gynogenesis. Journal of the Ghana Science Association Vol. 9 (2) 2007: pp. 32-37

The Referendum on Artificial Insemination

On 12 and 13 June 2005, the citizens of Italy were called to the polls to vote on law no. 40 of 19 February 2004, which for the first time in Italy established regulations on medically assisted reproduction (MAR). This law, even if it arrived later in Italy than in other European countries, was not suddenly “born.” Its adoption came about after a long and tormented debate that concluded with the approval of a text supported by a majority that crossed the boundary between the two political coalitions, which included almost the whole of the center-right and the majority of the Margherita. The four referendum questions, proposed by representatives of the left and supported by the Radical movement, aimed to introduce modifications to the law, which, stated simply, would allow research on surplus embryos for therapeutic purposes, genetic diagnosis pre-implantation, and the use of heterologous fertilization (i.e., donated sperm). All of these procedures are forbidden by law no. 40.

Superoxide dismutase affects the viability of thawed European mouflon (Ovis g. musimon) semen and the heterologous fertilization using both IVF and intracytoplasmatic sperm injection.

This study evaluated the effects of superoxide dismutase (SOD) on viability and acrosome integrity of European mouflon spermatozoa after cryopreservation and on the fertilization rates of sheep oocytes after i.v.f. or intracytoplasmatic sperm injection (i.c.s.i.). Frozen semen was thawed and washed with synthetic oviduct fluid supplemented with 0.6% bovine serum albumin. After centrifugation, the spermatozoa pellet was split into two culture systems: (i) without SOD; and (ii) in the presence of 1500 IU mL(-1) SOD. Sperm viability and acrosome integrity were evaluated simultaneously, immediately after thawing and after 3, 6 and 9 h of culture (5% CO2, 39 degrees C, 90% humidity), by incubating sperm with propidium iodide and fluorescein isothiocyanate-labelled Pisum sativum agglutinin. At the same time, sperm were assessed for motility using a standard scoring system (independent operators' observation of sperm) that graded degree of motility (i.e. 1 = immotile to 10 = maximum motility, as observed at the moment of thawing). For i.v.f., frozen-thawed semen derived from the two culture systems was placed in culture together with in vitro-matured sheep oocytes. For i.c.s.i., semen derived from the same culture systems as that for i.v.f. was used, and incubated for 1 h under standard conditions. The results showed a marked difference (P < 0.01) between the percentages of live spermatozoa in medium with SOD and those obtained in medium alone, after 3, 6 and 9 h of culture. The percentages of intact acrosome spermatozoa were higher in medium with SOD after 6 h (P = 0.05) of culture. Spermatozoa motility decreased significantly in SOD containing medium at 3 and 6 h of culture compared with motility in control medium. Fertilization rates were significantly lower in medium with SOD than in medium alone, whereas in the i.c.s.i. system fertilization rates were significantly higher in the presence of SOD. The results indicate that the addition of SOD to the culture media enhances the viability rates and the acrosome integrity of cryopreserved mouflon spermatozoa.

Mechanism of the block to hybridization and selfing between the sympatric ascidians Ciona intestinalis and Ciona savignyi.

The solitary ascidians Ciona intestinalis and Ciona savignyi co-occur in southern California harbors, but no hybrids have been recognized in nature. Numerous differences in their egg morphology were detected. Homologous (normal outcross) fertilization yielded 96-99% cleavage, where autologous (self) fertilization showed 3% and heterologous (hybrid) fertilization showed 0-1%. Acid treatment (pH 3.2) removed the block to selfing (P < 0.0001) but not hybridization for both species. Heterologous sperm bind to the vitelline coat (VC), but fail to penetrate. Enzymatic removal of the VC resulted in 91-97% cleavage with autologous and heterologous sperm (P < 0.0001). The vitelline coats of the two species differ in lectin binding to surface glycosides. Fertilization in both species is significantly inhibited by the lectins, fucose binding protein (P < 0.0001) and concanavalin A (P < 0.0001), and wheat germ agglutinin inhibits fertilization in C. intestinalis (P < 0.0001) but is without effect on C. savignyi fertilization. Self and hybrid blocks employ different mechanisms including glycoside composition and acid sensitivity.

Amphibian cross-fertilization and polyspermy.

A string of oocytes from Bufo arenarum (Ba) was inseminated with spermatozoa from Ba, Leptodactylus chaquensis (Lch), or Bufo paracnemis (Bp). Homologous insemination resulted in monospermic eggs and normal development, while in the case of heterologous fertilization both polyspermy and abnormal development were the rule. Oocytes from Ba inseminated with homologous spermatozoa, when reinseminated 30 sec after the first insemination with heterologous spermatozoa, exhibit a high rate of polyspermy and abnormal development. A lower rate of abnormalities was observed when reinsemination was carried out 60 sec after the first insemination. Normal development and an absence of polyspermy were observed in the case of eggs reinseminated 300 sec after initial insemination. This result indicated that the electrophysiological blockage of fertilization either was not triggered or was ineffective. The establishment of the permanent blockage of polyspermy, either by artificial activation of oocytes or as a result of their incubation with the products of the cortical granules, inhibited penetration of Ba eggs by heterologous sperm.

Effect of the donor oocyte breed on in vitro fertilization results in cattle

The effect of the cumulus on in vitro fertilization in bovines was examined. Follicular oocytes were cultured in medium 199 plus OCS and extra granulosa cells. Frozen-thawed bovine spermatozoa was separated by the swim-up technique, suspended in Talp medium and capacitated with heparin. Fresh sheep and goat semen was incubated for 4 h at room temperature, washed and spermatozoa were then suspended in Talp medium and capacitated by incubation at 38.5 °C and 5% CO2 in air and heparin.In experiment 1, cumulus-enclosed oocytes, denuded oocytes and denuded oocytes plus additional cumulus cells were incubated with a reduced concentration of bovine spermatozoa for 8 or 18 h. In Experiment 2, cumulus enclosed and denuded oocytes were incubated with bovine spermatozoa for 4, 6, 8 and 18 h using a sperm concentration adjusted to secure high fertilization rates. In Experiment 3, cumulus-enclosed and denuded bovine oocytes were incubated with either sheep or goat spermatozoa for 18 h. Fertilization rates were then calculated and compared statistically. The results showed that 1) the cumulus improved the fertilization rate only when cumulus cells were associated with the oocytes 2) the timing of sperm penetration was not modified by the cumulus and started at 4 h after sperm incubation and 3) the presence of the cumulus improved the heterologous fertilization rate only when sheep spermatozoa were used. The results suggest that the cumulus improves fertilization rate by providing a capacitation-inducing mechanism and by facilitating the interaction between capacitated spermatozoa and the zona pellucida surface.

Effect of the cumulus on in vitro fertilization of bovine matured oocytes

The effect of the cumulus on in vitro fertilization in bovines was examined. Follicular oocytes were cultured in medium 199 plus OCS and extra granulosa cells. Frozen-thawed bovine spermatozoa was separated by the swim-up technique, suspended in Talp medium and capacitated with heparin. Fresh sheep and goat semen was incubated for 4 h at room temperature, washed and spermatozoa were then suspended in Talp medium and capacitated by incubation at 38.5 °C and 5% CO 2 in air and heparin. In experiment 1, cumulus-enclosed oocytes, denuded oocytes and denuded oocytes plus additional cumulus cells were incubated with a reduced concentration of bovine spermatozoa for 8 or 18 h. In Experiment 2, cumulus enclosed and denuded oocytes were incubated with bovine spermatozoa for 4, 6, 8 and 18 h using a sperm concentration adjusted to secure high fertilization rates. In Experiment 3, cumulus-enclosed and denuded bovine oocytes were incubated with either sheep or goat spermatozoa for 18 h. Fertilization rates were then calculated and compared statistically. The results showed that 1) the cumulus improved the fertilization rate only when cumulus cells were associated with the oocytes 2) the timing of sperm penetration was not modified by the cumulus and started at 4 h after sperm incubation and 3) the presence of the cumulus improved the heterologous fertilization rate only when sheep spermatozoa were used. The results suggest that the cumulus improves fertilization rate by providing a capacitation-inducing mechanism and by facilitating the interaction between capacitated spermatozoa and the zona pellucida surface.

Cytogenetic studies in motile sperm from normal men.

Most studies on human sperm chromosomes from normal men involve the heterologous fertilization of zona free hamster eggs by unselected human sperm. In this work, we have performed cytogenetic studies of highly motile sperm, selected by a swim-up method. A total of 505 motile human sperm complements from three normal donors was analysed. The total frequency of sperm with chromosomal abnormalities (10.9%; 6.9% structural aberrations and 4.0% aneuploidy) and the sex ratio (50.4% X:49.6% Y) were similar to those obtained from whole semen samples. Our results indicate that the selection of motile sperm does not imply chromosomal selection.

The gamete membrane fusion test to assay the fertilizing ability of human spermatozoa.

The value of the gamete fusion test for the assessment of human spermatozoa is assessed. The factors influencing the test are discussed, including the conditions of heterologous fertilization using zona-free oocytes such as the nature of sperm preparation, sperm concentration and capacitation time, and the importance of albumin in the medium. The oocytes can be examined before or after fixation, and the spermatozoa around the eggs assessed for acrosome changes, while the number of sperm tails on the oocyte can be related to the number of chromatin decondensations. The test provides information on the fertilizing ability of spermatozoa of given individuals, and may prove of value in testing contraceptives.

Experimental studies ofStrongylocentrotus droebachiensisandS. pallidus

Abstract The problem as to whether Strongylocentrotus pallidus (G. O. Sars, 1871) is a distinct species or only a colour-variety of S. droebachiensis was subjected to a renewed investigation. For the first time simultaneous experiments on homologous and heterologous fertilization of S. droebachiensis and S. pallidus were carried out. These experiments revealed the existence of clear specificity barriers between the two forms. Studies of mitosis in hybrid embryos indicated that chromosomal aberrations are frequent. Electron microscopic investigations demonstrated that the ultra-structure of the cortical granules is different in the two species. The differences observed supply strong evidence in support of the opinion that Strongylocentrotus pallidus is a good species.

The influence of the jelly coat in situ and in solution on cross fertilization in sea urchins

The jelly coat represents in Psammechinus microtuberculatus and Echinocardium cordatum a strong barrier to heterologous fertilization. The inhibiting action on fertilization exerted by jelly solutions is not species-specific. Very dilute jelly solutions (= egg water) enhance the fertilization rate of jelly-free eggs inseminated with heterologous sperm. This effect of jelly solution does not seem to be species-specific. The addition of dilute jelly solution to eggs with intact jelly coats and inseminated with homologous or heterologous sperm does not markedly influence the fertilization rate. This is probably explained by the fact that the eggs are already surrounded by “egg water” (= the jelly coat). Heparin was found to exert an action similar to that of jelly solution. Heparin and jelly solution are only effective when present at the time of insemination and they both increase the motility of the spermatozoa.