Oocytes undergo numerous biochemical and morphological changes during their development from preantral to preovulatory phases. In vitro studies have suggested several compounds that might induce oocyte maturation. Heparin is a natural component of ooplasm, follicular fluid and uterine fluid and previous studies indicated that it might act as a chromatin maturation factor in bovine oocytes. We tested this hypothesis in vitro by timing germinal vesicle breakdown (GVBD) and first polar body (PB) formation without any other natural or introduced factors that might influence the rate of oocyte maturation. We also determined if these oocytes could be fertilized.

Bovine oocytes were incubated in a salt medium and TCM 199 supplemented with different concentrations of heparin for 24 h at 37.5 °C in a humidified atmosphere of 5% CO2. With 1.0 and 6.5 mg/ml heparin, the time of GVBD was reduced from 4.7 ± 1.1 h to about 1.5 h and the time of first PB formation was reduced from 22.0 ± 1.1 h to 9.0–11.0 h in salt medium. In TCM 199, only 6.5 mg/ml heparin significantly reduced the time of PB formation. In both incubation media, 1.0 and 6.5 mg/ml heparin induced GVBD, extrusion of the first PB and formation of the metaphase II nucleus. Moreover, heparin did not interfere with the fertilization of oocytes matured in TCM 199. Based on the results, we propose that heparin plays an important role in the rearrangement of the oocyte chromatin and acts as an oocyte maturation factor.

Careful attention has been focused recently on DNA quality in human IVF. Therefore a variety of methods has been developed to evaluate DNA integrity, especially concerning fragmentation. Using liquid chromatography and mass spectrometry (LC/MS/MS) for our best sperm samples, we have established reference values for several oxidative lesions, in order to gain insights into the cause of DNA lesions. Besides 8-oxodeoxyguanosine, we found rather high levels of two ethenonucleosides: 1,N6-ethenoadenosine and 1,N2-ethenoguanosine. These compounds probably arise from a reaction with 4-hydroxy-2-nonenal, the main aldehyde compound released during lipid peroxidation, or after occupational exposure to vinyl chloride. The quantity of chlorinated bases detected is low. All of this decay has to be repaired by the oocytes at the time of fertilization or immediately after. This aspect should not be overlooked in assisted reproductive technology, in order to understand risks and limitations.

In this study, nuclear transfer (NT) embryos were produced by using C57Bl/6 mouse morula blastomeres and Kunming mouse metaphase II (MII) oocytes as donors and recipients, respectively, to investigate the effects of sucrose treatment of MII oocytes with different concentrations on the manipulation time of NT, electrofusion and the in vitro and in vivo development of reconstructed embryos. The results demonstrated that: (i) when the oocytes were enucleated with 1, 2 and 3% sucrose treatment, respectively, the enucleating rates were not affected by the different sucrose concentrations, but the manipulation time had significant difference and the mean nuclear transfer manipulation times of every oocyte were 180 ± 10 s, 130 ± 10 s and 120 ± 10 s, respectively; (ii) different sucrose concentrations had no significant effects on the fusion rate and the in vitro developmental potential of the NT embryos (p > 0.05). Furthermore, 59 embryos were transplanted into the oviducts of two recipients. In the end, three dead full-term developed fetuses were obtained on 21 days post coitus (dpc). These results suggested that the mouse MII oocytes enucleated via sucrose treatment might be an alternative source for mouse cloning and could support the embryonic NT embryos developed to term in vivo.

Early studies have shown that some mouse cumulus–oocyte complexes (COCs) stored at room temperature for 24 h still retained full developmental potential. In this study, we stored denuded mouse oocytes (DOs) at room temperature (25 °C) for 24 h and activated these oocytes with 10 mM SrCl2 or fertilized the oocytes by IVF. We found that nearly half of the DOs stored at room temperature for 1 day can be fertilized normally by IVF and that two foster mothers gave birth to seven pups. Embryos from stored oocytes were cultured in CZB medium with or without 1 μg/ml 17β-estradiol (E2). The numbers of embryo that developed to morula/blastocyst stage after parthenogenetic activation and IVF were significantly increased when E2 was added to the culture (p < 0.05). These results suggest that E2 might improve mouse embryo development in vitro. The birth of seven agouti pups and their healthy growth indicated that the storage of DOs at room temperature for 1 day may be a practical procedure for mammalian reproduction.

The objective of this article was to estimate quantitative differences for GAPDH transcripts and poly(A) mRNA: (i) between oocytes collected from cumulus–oocyte complexes (COCs) qualified morphologically as grades A and B; (ii) between grade A oocytes before and after in vitro maturation (IVM); and (iii) among in vitro-produced embryos at different developmental stages. To achieve this objective a new approach was developed to estimate differences between poly(A) mRNA when using small samples. The approach consisted of full-length cDNA amplification (acDNA) monitored by real-time PCR, in which the cDNA from half of an oocyte or embryo was used as a template. The GAPDH gene was amplified as a reverse transcription control and samples that were not positive for GAPDH transcripts were discarded. The fold differences between two samples were estimated using delta Ct and statistical analysis and were obtained using the pairwise fixed reallocation randomization test. It was found that the oocytes recovered from grade B COCs had quantitatively less poly(A) mRNA (p < 0.01) transcripts compared with grade A COCs (1 arbitrary unit expression rate). In the comparison with immature oocytes (1 arbitrary unit expression rate), the quantity of poly(A) mRNA did not change during IVM, but declined following IVF and varied with embryo culture (p < 0.05). Amplification of cDNA by real-time PCR was an efficient method to estimate differences in the amount of poly(A) mRNA between oocytes and embryos. The results obtained from individual oocytes suggested an association between poly(A) mRNA abundance and different morphological qualities of oocytes from COCs. In addition, a poly(A) mRNA profile was characterized from oocytes undergoing IVM, fertilization and blastocyst heating.

Hyaluronan (HA) is glycosaminoglycan that is present from the start of embryonic development and its role and concentration increases with embryo development. The objective of this study was to evaluate if the presence of HA in TCM-199 culture medium had an effect on the development and quality of bovine embryos. There was no effect of HA on the total number of zygotes developing to blastocysts on day 7, however more expanded and hatched blastocyst stages were observed on days 8 and 9 in the group supplemented with HA (p < 0.05). Following freeze/thawing, significantly more (p < 0.05) embryos cultured in medium supplemented with HA hatched than those cultured in TCM-199 alone or those with BSA. Medium supplemented with HA and BSA significantly increased the level of expression of glucose metabolism Glut-1 gene and embryo compaction Cx43 gene (p < 0.05), and had no effect on Glut-5 and IGF-II expression. In addition, HA presence in culture decreased the level of expression of apoptosis Bax and oxidative stress SOX genes (p < 0.05). There was significant difference in total number of nuclei between TCM-199 medium only and the remaining media containing BSA or HA plus BSA, between which there was no difference. In summary, our results indicate that the addition of high molecular weight HA to TCM-199 medium that contains BSA on day 4 of culture improved embryo development to hatching and hatched blastocysts and the quality of produced embryos, which were superior to embryos cultured without HA addition.

The positive effect of strontium ions (Sr2+) on sperm motility, capacitation and acrosome reaction has been demonstrated in the mouse, human, guinea pig and hamster. In the present study, we have evaluated the effect of Sr2+ on the viability and acrosome morphology of boar spermatozoa, and on the fertilization and development after the microinjection of Sr2+-treated spermatozoa into porcine oocytes. Before incubation, 79% of spermatozoa were classified as propidium iodide (PI)-negative (live) using the LIVE/DEAD Sperm Viability Kit. After incubation with strontium chloride (SrCl2), 39% (0 mM; no divalent cations), 25% (1.9 mM) and 24% (7.5 mM) of them were classified as PI-negative. The proportion of spermatozoa that had initiated the acrosome reaction was higher in Sr2+-containing medium than in Sr2+-free medium, when assessed by electron microscopy. There was no significant difference in percentage of spermatozoa initiating the acrosome reaction between Sr2+-treated groups (1.9 mM: 22%, 7.5 mM: 33%, p > 0.05). After the microinjection of spermatozoa incubated with SrCl2, 67% (1.9 mM) and 61% (7.5 mM) of injected oocytes were successfully fertilized, and then 43% (1.9 mM) and 41% (7.5 mM) contained a fully decondensed sperm head. Sham-injected oocytes were significantly activated at a lower rate than Sr2+-treated groups (27%, p < 0.05). Next, after microinjection of spermatozoa incubated with 1.9 mM SrCl2 (n = 51), 45% of injected oocytes cleaved on day 2, and 18% developed to blastocysts on day 7 (sham-injection, n = 48: 15% to cleavage and 0% to blastocyst). These results demonstrate that Sr2+ is likely to positively affect the fertilizing capacity of spermatozoa in the pig.

The present study examines the use of buffalo preantral follicles as a source of oocytes for in vitro embryo production. Preantral follicles were isolated from abattoir-derived buffalo ovaries and were grown for 100 days in five different culture systems: (1) minimum essential medium (MEM); (2) coconut water; (3) MEM + ovarian mesenchymal cell (OMC) co-culture; (4) MEM + granulosa cell (GC) co-culture; or (5) MEM + cumulus cell (CC) co-culture. Low growth rates for the preantral follicles were observed when follicles were cultured in MEM or coconut water medium. Moderate growth rates were seen for OMC and GC co-cultures, and high rates of growth were observed when follicles were grown in CC co-culture. The survival of preantral follicles was low in the MEM culture (<25%), but was over 75% in the other culture systems. Oocytes were not recovered from the MEM group, while an oocyte recovery rate of 80–100% was observed when the follicles were cultured with coconut water/somatic cells. Transferable embryos could be produced only with the oocytes obtained from preantral follicles grown in the OMC and CC co-culture systems. This study demonstrates, for the first time, that it is possible to produce buffalo embryos by in vitro fertilization of oocytes derived from in vitro grown preantral follicles.

In this study we investigated ovulation in vitro using ovary samples from Bufo arenarum with respect to their response to stimulation with homologous pituitary homogenate (HPH) or with progesterone and prostaglandins (PGF2α and PGE1) as intermediates of pituitary action. Ovary samples were obtained from animals captured during the breeding period. Our results demonstrate that the ovulatory response to all different inducers was dose dependent, the highest percentage of ovulated oocytes being obtained with HPH treatment. An important increase in the ovulatory response was obtained by the association of PGF2α with either HPH or progesterone at suboptimal doses, indicating that this prostaglandin induced a synergistic potentiating effect. Incubation with cyclooxygenase inhibitors (indomethacin or diclofenac sodium) produced a significant decrease in the ovulation induced by HPH, demonstrating that prostaglandins are involved in the action of the pituitary gland in this process. According to our results, PGE1 not only had no participation in the ovulatory process, but also produced an inhibitory effect on ovulation induced by HPH treatment.

The 330 kDa fibrillar glycoprotein hyalin is a well known component of the sea urchin embryo extracellular hyaline layer. Only recently, the main component of hyalin, the hyalin repeat domain, has been identified in organisms as widely divergent as bacteria and humans using the GenBank database and therefore its possible function has garnered a great deal of interest. In the sea urchin, hyalin serves as an adhesive substrate in the developing embryo and we have recently shown that exogenously added purified hyalin from Strongylocentrotus purpuratus embryos blocks a model cellular interaction in these embryos, archenteron elongation/attachment to the blastocoel roof. It is important to demonstrate the generality of this result by observing if hyalin from one species of sea urchin blocks archenteron elongation/attachment in another species. Here we show in three repeated experiments, with 30 replicate samples for each condition, that the same concentration of S. purpuratus hyalin (57 μg/ml) that blocked the interaction in living S. purpuratus embryos blocked the same interaction in living Lytechinus pictus embryos. These results correspond with the known crossreactivity of antibody against S. purpuratus hyalin with L. pictus hyalin. We propose that hyalin–hyalin receptor binding may mediate this adhesive interaction. The use of a microplate assay that allows precise quantification of developmental effects should help facilitate identification of the function of hyalin in organisms as divergent as bacteria and humans.

Local application of the agonists of serotonin receptors of third type 5-HTQ, SR57277A and quipazine into the interblastomere cleft of the sea urchin Paracentrotus lividus embryo during first cleavage division, evokes specific membrane currents, whereas application of these drugs out of contact area show currents of lower amplitude and longer latent period. At the same time 5-HT3-receptor agonist quipazine imitates interblastomere signal in half embryos, but corresponding antagonists prevent it. Present data develop the hypothesis of protosynapse, demonstrating that the distribution of membrane serotonin receptors is limited to the period of cleavage division and localized in the interblastomere contact area. A possible role of spatial–temporal restriction of receptors at the interblastomere contact area is discussed in relation to the subsequent embryo development.

The objective of this study was to compare developmental capacity of rabbit chimeric embryos and the allocation of the EGFP gene expression to the embryoblast (ICM) or embryonic shield. We produced chimeric embryos (TR<>N) by synchronous transfer of two or three blastomeres at the 16-cell stage from transgenic (TR) into normal host embryos (N) at the same stage. In the control group, two to three non-transgenic blastomeres were used to produce chimeric embryos. The TR embryos were produced by microinjection of EGFP into both pronuclei of fertilized rabbit eggs. The developmental rate and allocation of EGFP-positive cells of the reconstructed chimeric embryos was controlled at blastocyst (96 h PC) and embryonic shield (day 6) stage.

All chimeric embryos (120/120, 100%) developed up to blastocyst stage. Using fluorescent microscope, we detected green signal (EGFP expression). In 90 chimeric (TR<>N) embryos (75%). Average total number of cells in chimeric embryos at blastocyst stage was 175 ± 13.10, of which 58 ± 2.76 cells were found in the ICM area. The number of EGFP-positive cells in the ICM area was 24 ± 5.02 (35%). After the transfer of 50 chimeric rabbit embryos at the 16-cell stage, 20 embryos (40%) were flushed from five recipients on day 6 of pregnancy, of which five embryos (25%) were EGFP positive at the embryonic shield stage.

Our results demonstrate that transgenic blastomeres in synchronous chimeric embryos reconstructed from TR embryos have an ability to develop and colonize ICM and embryonic shield area.