We studied whether bovine embryos developing after in vitro fertilization (IVF) with sex-sorted spermatozoa differed in developmental kinetics, quality and sex ratio from embryos produced with unsorted spermatozoa. Abattoir-derived oocytes were fertilized with X-sorted, Y-sorted or unsorted spermatozoa from a single bull. To evaluate economical use of the sex-sorted spermatozoa, washed spermatozoa from a single straw (2 million spermatozoa) were used to fertilize each batch of collected oocytes without any further isolation steps. Concentration of the unsorted spermatozoa was adjusted accordingly. Fertilizations were assessed by staining sperm asters at 10 hpi and pronuclei at 20 hpi. Embryo development and morphological quality were monitored on days 2, 7, 8 and 9 of the development (IVF = day 0). All embryos were sexed using PCR. Following fertilization, penetration and subsequent cleavage rates were compromised in the X-sorted group compared with the Y-sorted and unsorted groups (penetration: 58.0% vs. 89.8% and 90.0%, cleavage: 65.3% vs. 81.5% and 75.0%). The use of the sex-sorted spermatozoa did not, however, reduce the proportion of transferable embryos (sex-sorted 29.6% vs. unsorted 27.7%) or their quality (quality 1: sex-sorted 36.0% vs. unsorted 19.9%). The Y-sorted spermatozoa produced more transferable embryos of better quality than the X-sorted spermatozoa (days 7–8: 31.9% vs. 26.4%, quality 1: 38.9% vs. 30.6%). On average, out of 10 transferable embryos, nine were of the predicted sex in the X- and Y-sorted spermatozoa groups. These results indicate that low numbers of X- and Y-sorted spermatozoa can be used successfully for female and male embryo production in vitro.

Tetraspanin CD9 is the only protein of the oocyte membrane (oolemma) known to be required for the fusion of gametes during fertilization in the mouse. Using electron microscopy and immunostaining we examined the differences in localization of CD9 between ovulated oocytes, zygotes and parthenogenetically activated eggs (parthenogenotes). Changes in ultrastructure of oolemma, which take place in oocytes after fertilization or artificial activation, were also assessed. We demonstrated that after fertilization the level of CD9 present on microvilli of zygote was two times lower than its level on the oolemma of the oocyte. In addition, we showed that the distribution of microvilli is less uniform in the zygotes than in the unfertilized oocytes. We propose that the changes of microvilli distribution and their CD9 content are responsible for the development of the oocyte membrane block to sperm penetration.

The aim of our study was to analyse chromosomal aneuploidy occurence in rabbit oocytes and embryos. Chromosomal analysis was done in rabbit oocytes and rabbit preimplantation embryos at 2-, 4- and 8-cell stages derived from in vivo fertilization. For mitotic cycle synchronization at the metaphase stage, 2-, 4- and 8-cell embryos were incubated in k-DMEM medium, supplemented with colcemid (1 μg/ml), for 7, 12 or 13h respectively. Success of metaphase synchronization was at values of 100, 86.1 and 92.2% for 2-, 4- and 8-cell embryos respectively. Recovery rate of analysable metaphase plates was reached at 58.8%, 83.9% and 59.8% for 2-, 4- and 8-cell embryos and 100% for oocytes. Significant difference (p < 0.01) in aneuploidy rate between oocytes (40.7%) and 2-cell embryos (62.5%) was found. These results demonstrate higher efficiency of synchronization of embryo cells at the metaphase stage, what may contribute to elevating the proportion of analysable nuclei.

The objective of this work was to evaluate the selection of immature bovine oocytes by brilliant cresyl blue dye (BCB) and expression of transcripts MATER and ZAR1. Cumulus–oocyte complexes (COCs) from slaughterhouse ovaries were exposed to BCB diluted in mDPBS and incubated for 60 min at 38.5 °C in humidified air. After exposure those COCs were distributed in two groups, according to their cytoplasm colour: BCB+ (coloured cytoplasm) or BCB− (colourless cytoplasm). The control group was submitted to in vitro maturation (IVM) immediately after morphological selection and holding control group COCs were exposed to mDPBS without BCB but in the same incubation conditions of BCB+ and BCB− group. The COCs of all groups were submitted to IVM, in vitro fertilization (IVF) and in vitro culture (IVC). Cleavage rate (72 h post-insemination) was similar between control (65.3%) and BCB+ (64.4%) groups, but greater than (p < 0.05) holding control (49.8%) and BCB− (51.3%) groups. Blastocyst rate (192 h post-insemination) was not different between BCB+ (18.5%) and control (16.3%) groups, but greater (p < 0.05) than BCB− (8.4%) group. No difference was found for blastocyst rate between holding control group (14.2%), control and BCB+ groups. The relative expression of MATER and ZAR1 genes was evaluated by real-time PCR in immature oocytes collected from the control, holding control, BCB+ and BCB− groups. Despite the relative expression of MATER in holding control, BCB+ and BCB− were down regulated in comparison to control group there was no statistical difference (p > 0.05) in the relative expression of MATER and ZAR1 transcripts among groups. The results indicate that the BCB dye detects immature oocyte populations with different developmental competence, although no improvement in in vitro embryo production using oocytes exposed or not to BCB was observed. Development competence of immature oocytes exposed to BCB does not seem to be associated with variations in the expression of MATER and ZAR1 transcripts.

The guinea pig spermatozoid nucleus contains actin, myosin, spectrin and cytokeratin. Also, it has been reported that phalloidin and/or 2,3-butanedione monoxime retard the sperm nuclear decondensation caused by heparin, suggesting a role for F-actin and myosin in nuclear stability. The presence of an F-actin/myosin dynamic system in these nuclei led us to search for proteins usually related to this system. In guinea pig sperm nuclei we detected calmodulin, F-actin, the myosin light chain and an actin-myosin complex. To define whether calmodulin participates in nuclear-dynamics, the effect of the calmodulin antagonists W5, W7 and calmidazolium was tested on the decondensation of nuclei promoted by either heparin or by a Xenopus laevis egg extract. All antagonists inhibited both the heparin- and the X. laevis egg extract-mediated nuclear decondensation. Heparin-mediated decondensation was faster and led to loss of nuclei. The X. laevis egg extract-promoted decondensation was slower and did not result in loss of the decondensed nuclei. It is suggested that in guinea pig sperm calmodulin participates in the nuclear decondensation process.

Follicle stimulating hormone (FSH) and luteinizing hormone (LH) are commonly added to maturation media to improve cumulus expansion known to be a predictor of oocyte maturation. Therefore, effects of various concentrations of FSH (1000 ng/ml), LH (1000 ng/ml) and FSH + LH (1000 ng/ml each) in comparison with control (without FSH + LH) cultured oocytes were investigated. FSH and LH (1000 ng/ml each) induced significantly more cumulus expansion and polar body numbers, as compared with control and treatments of 1000 ng/ml FSH and 1000 ng/ml LH alone. Expression of FSH receptor (r), LHr and Cx43 mRNAs was determined by real-time PCR in cumulus–oocyte complexes (COCs) and denuded oocytes at different maturation times. Expression of all three genes was higher in COCs compared with denuded oocytes, confirming the importance of cumulus cells in oocyte maturation. FSHr and connexin 43 (Cx43) mRNA abundance in both COCs and denuded oocytes was highest at 0–6 h of maturation and decreased subsequently. However, LHr mRNA abundance increased from 6 h up to 24 h of maturation. The study concluded that FSH, LH receptors and Cx43 gene expression regulation is an index related to oocyte maturation.

In this paper we assessed: (i) the change in nitric oxide synthase (NOS) isoforms' expression and intracellular localization and in NOS mRNA in porcine oocytes during meiotic maturation; (ii) the effect of NOS inhibition by Nω-nitro-l-arginine methyl ester (l-NAME) and aminoguanidine (AG) on meiotic maturation of cumulus–oocyte complexes (COC) as well as denuded oocytes (DO); and (iii) nitric oxide (NO) formation in COC. All three NOS isoforms (eNOS, iNOS and nNOS) and NOS mRNA (eNOS mRNA, iNOS mRNA and nNOS mRNA) were found in both porcine oocytes and their cumulus cells except for nNOS mRNA, which was not detected in the cumulus cells. NOS isoforms differed in their intracellular localization in the oocyte: while iNOS protein was dispersed in the oocyte cytoplasm, nNOS was localized in the oocyte cytoplasm and in germinal vesicles (GV) and eNOS was present in dots in the cytoplasm, GV and was associated with meiotic spindles. l-NAME inhibitor significantly suppressed metaphase (M)I to MII transition (5.0 mM experimental group: 34.9% MI, control group: 9.5% MI) and at the highest concentration (10.0 mM) also affected GV breakdown (GVBD); in contrast also AG inhibited primarily GVBD. The majority of the oocytes (10.0 mM experimental group: 60.8%, control group: 1.2%) was not able to resume meiosis. AG significantly inhibited GVBD in DO, but l-NAME had no significant effect on the GVBD of these cells. During meiotic maturation, NO is formed in COC and the NO formed by cumulus cells is necessary for the process of GVBD.

The development of preservation techniques for male gametes at room temperature might allow us to store them in a simple and cost-effective manner. In this study, we studied the use of pure salt or sugar to preserve the whole cauda epididymidis, because it is known that food can be preserved in this way at room temperature for long periods. Mouse epididymides were placed directly in powdered salt (NaCl) or sugars (glucose or raffinose) for 1 day to 1 year at room temperature. Spermatozoa were recovered from the preserved organs after being rehydrated with medium and then isolated sperm heads were microinjected into fresh oocytes. Importantly, the oocyte activation capacity of spermatozoa was maintained after epididymal storage in NaCl for 1 year, whereas most untreated spermatozoa failed to activate oocytes within 1 month of storage. Pronuclear morphology, the rate of extrusion of a second polar body and the methylation status of histone H3 lysine 9 (H3K9me3) in those zygotes were similar to those of zygotes fertilized with fresh spermatozoa. However, the developmental ability of the zygotes decreased within 1 day of sperm storage. This effect led to nuclear fragmentation at the 2-cell embryo stage, irrespective of the storage method used. Thus, although the preserved sperm failed to allow embryo development, their oocyte activation factors were maintained by salt storage of the epididymis for up to 1 year at room temperature.

It is well known that melatonin provokes reproductive alterations in response to changes in hours of daylight in seasonally breeding mammals, exerting a regulatory role at different levels of the hypothalamic–pituitary–gonadal axis. Although it has also been demonstrated that melatonin may affect testicular activity in vertebrates, until now, very few data support the hypothesis of a local action of melatonin in the male gonads. The aim of this study was to investigate whether MT1, MT2 melatonin receptors and the H9 melatonin-related receptor, are expressed in the adult rat testes and during development. A semi-quantitative RT-PCR method was used to analyse the expression of MT1, MT2 and H9 receptors mRNAs in several rat tissues, mainly focusing on testes during development and adult life. Our results provide molecular evidences of the presence of both MT1 and, for the first time, MT2 melatonin receptors as well as of the H9 melatonin-related receptor in the examined tissues, including adult testes. During development MT1 and MT2 transcripts are expressed at lower levels in testes of rats from 1 day to 1 week of age, lightly increased at 2 weeks of age and remained permanently expressed throughout development until 6 months. These data strongly support the hypothesis that melatonin acts directly in male vertebrate gonads suggesting that rat testes may be a suitable model to verify the role of indolamine in vertebrate testicular activity.

Malondialdehyde (MDA), a product involved in membrane lipid peroxidation, was dosed in the sperm of 163 patients who had consulted the clinic regarding hypofertility. We attempted to determine if there was correlation between MDA content, sperm World Health Organization parameters and DNA fragmentation that results mainly from reactive oxygen species assaults. We found that no correlation could be established; however MDA and sperm decondensation were shown to be significantly linked. The impact of membrane polyunsaturated fatty acids and the role of phospholipid hydroperoxide glutathione peroxidase are discussed.

In cattle, embryos elongate before implantation and after hatching. Changes in gene expression during this transition are not well studied. Especially important are variations in the expression of pluripotency-associated genes as a result of assisted reproductive biotechnologies, such as cloning and in vitro fertilization (IVF). We hypothesize that there will be a decline in the expression of key pluripotency-associated genes and an increase in the expression of IFN-τ in elongated embryos when compared with day-7 blastocysts. To test this we generated cloned and IVF bovine day-7 blastocyst and day-17 elongated embryos (day 0 = day of nucleus transfer or IVF). Gene expression in all embryos was assessed via RT-qPCR. OCT4 was overexpressed (p < 0.05) in the cloned blastocysts when compared with IVF. No differences in gene expression at this stage between cloned and IVF embryos were found for EOMES, NANOG and FGF4. At elongation EOMES, NANOG and FGF4 were upregulated in IVF embryos (p < 0.05). IFN-τ and OCT4 were expressed at similar levels. There were changes in the expression levels for all transcripts between blastogenesis and elongation. NANOG, IFN-τ and EOMES were overexpressed in all the elongated embryos (p < 0.05), FGF4 was underexpressed in both treatments. OCT4 dropped drastically in the cloned elongated embryos, but not in the IVF. Interestingly only adult donor cells (but not fetal) from which the cloned embryos originated also expressed high levels of OCT4. Our findings might help to understand the shift of gene expression during elongation and to identify key markers of embryonic development useful for embryo screening purposes.