Repeats as global DNA methylation marker in bovine preimplantation embryos

DNA methylation undergoes dynamic changes and is a crucial part of the epigenetic regulation during mammalian early development. To determine the DNA methylation levels in bovine embryos, we applied a bisulfite sequencing based method aimed at repetitive sequences including three retrotransposons (L1_BT, BovB, and ERV1-1-I_BT) and Satellite I. A more accurate estimate of the global DNA methylation level compared to previous methods using only one repeat sequence, like Alu, could be made by calculation of the weighted arithmetic mean of multiple repetitive sequences, considering the copy number of each repetitive sequence. Satellite I and L1_BT showed significant methylation reduction at the blastocyst stage, while BovB and ERV1-1-I_BT showed no difference. The mean methylation level of the repetitive sequences during preimplantation development was the lowest at the blastocyst stage. No methylation difference was found between embryos cultured in 5% and 20% O-2. Because mutations of CpGs negatively influence the calculation accuracy, we checked the mutation rate of the sequenced CpG sites. Satellite I and L1_BT showed a relatively low mutation rate (1.92 and 3.72% respectively) while that of ERV1-1-I_BT and BovB was higher (11.95 and 24% respectively). Therefore we suggest using a combination of repeats with low mutation rate, taking into account the proportion of each sequence, as a relatively quick marker for the global DNA methylation status of preimplantation stages and possibly also for other cell types

Influence of sperm-oocyte coincubation period on porcine in vitro fertilization (IVF) efficiency

A major obstacle for successful in vitro production of porcine embryos is the polyspermic fertilization. One possibility to reduce polyspermic penetration is decreasing the number of spermatozoa added to the fertilization medium. Unfortunately, the lower rate of polyspermy is accompanied by a reduced penetration rate. A short gamete coincubation period of 10 min has been described to obtain fertilization rates similar to 6 h of coincubation and may improve IVF efficiency (number of monospermic fertilized oocytes/total number inseminated) depending on sperm-oocyte ratio (Gil, 2007, Theriogenology, 67(3), 620–626). Here we demonstrate that the optimal coincubation period in our IVF conditions is between 10 min and 6 h. In vitro matured oocytes (n = 600) were inseminated with frozen-thawed epididymal semen with 600 spermatozoa per oocyte and coincubated for 2, 4 and 6 h. At 2 and 4 h post insemination (hpi), oocytes were vortexed and transferred to fertilization medium without spermatozoa. At 6 hpi, presumed zygotes of all groups were washed three times in culture medium and cultured. At 22 hpi, zygotes were fixed overnight and stained with Hoechst 33,342 for the assessment of fertilization and polyspermy. The IVF efficiency was higher for the 4 h group (40 ± 5%) than the 2 and 6 h group (19 ± 8% and 17 ± 5%). Between 4 and 6 h of gamete coincubation, the increase in the number of polyspermic oocytes was relatively higher than the increase in penetration rate (+39% vs. +15%), resulting in a decline in efficiency. (This study was supported by Research Foundation-Flanders)

Features of reproduction and assisted reproduction in the white (Ceratotherium simum) and black (Diceros bicornis) rhinoceros

Despite the worldwide increase of rhinoceros calf numbers, the growth of the population of white and black rhinoceros is slowing down mainly due to anthropogenic causes, such as poaching and habitat loss. Assisted reproduction is one of the methods of preserving the valuable genomes of these animals from being lost, and assists in breeding them in captivity to maintain the specie(s) numbers and provide an option for possible reintroduction into the wild. Since wild rhinoceros are difficult to handle and examine clinically, most of the current information available on their reproductive characteristics has been gained from captive rhinoceros populations. Nevertheless, very little is known about rhinoceros reproduction. Since the rhinoceros belongs to the odd-toed ungulates (Perissodactyls) group, like the horse and the tapir, the horse has been proposed as a suitable model to study reproduction and artificial reproductive techniques in the rhinoceros. In this review, the current knowledge of the reproduction of the rhinoceros is summarized

Hyperoestrogenisme bij een vrouwelijke chihuahuapup

A female intact Chihuahua pup was presented at the age of four months with complaints of vulvar swelling and precocious sexual behaviour. Both complaints started to develop at the age of 9-10 weeks. During clinical examination, inspection revealed swollen mammary glands. The differential diagnosis was gonadotrophin-independent (peripheral) precocious puberty (or precocious pseudopuberty) as a result of the exogenous intake of estrogens, or intersexuality. The first diagnosis was the most probable one since the female owner of the dog applied an estrogen gel on face and shoulders on a daily basis. The owner was advised to avoid contact of the pup with the gel. This resulted in a positive evaluation after two months, with a complete disappearance of the complaints four months after the first examination

The essence of fertilization: oocyte meets sperm

The problem of reduced fertility in high yielding dairy cattle is a very complicated one, and the relationship between various measures of fertility and level of milk production remains controversial. In this brief review the essence of the problem is considered: what is the oocyte's and the sperm's contribution, and what is the importance of the resulting embryo in the declining fertility of the Holstein Friesian cow

pH-dependent effects of procaine on equine gamete activation

Procaine directly triggers pH-dependent cytokinesis in equine oocytes and induces hypermotility in stallion spermatozoa, an important event during capacitation. However, procaine-induced hyperactivated motility is abolished when sperm is washed to remove the procaine prior to sperm-oocyte co-incubation. To understand how procaine exerts its effects, the external Ca2+ and Na+ and weak base activity dependency of procaine-induced hyperactivation in stallion spermatozoa was assessed using computer-assisted sperm analysis. Percoll-washed stallion spermatozoa exposed to Ca2+-depleted (+2 mM EGTA) procaine-supplemented capacitating medium (CM) still demonstrated hyperactivated motility, whereas CM without NaCl or Na+ did not. Both procaine and NH4Cl, another weak base, were shown to trigger a cytoplasmic pH increase (BCECF-acetoxymethyl (AM)), which is primarily induced by a pH rise in acidic cell organelles (Lysosensor green dnd-189), accompanied by hypermotility in stallion sperm. As for procaine, 25 mM NH4Cl also induced oocyte cytokinesis. Interestingly, hyperactivated motility was reliably induced by 2.5-10 mM procaine, whereas a significant cytoplasmic cAMP increase and tail-associated protein tyrosine phosphorylation were only observed at 10 mM. Moreover, 25 mM NH4Cl did not support the latter capacitation characteristics. Additionally, cAMP levels were more than 10x higher in boar than stallion sperm incubated under similar capacitating conditions. Finally, stallion sperm preincubated with 10 mM procaine did not fertilize equine oocytes. In conclusion, 10 mM procaine causes a cytoplasmic and acidic sperm cell organelle pH rise that simultaneously induces hyperactivated motility, increased levels of cAMP and tail-associated protein tyrosine phosphorylation in stallion spermatozoa. However, procaine-induced hypermotility is independent of the cAMP/protein tyrosine phosphorylation pathway

Porcine oocyte maturation in vitro : role of cAMP and oocyte-secreted factors: a practical approach

Polyspermy or the penetration of more than one sperm cell remains a problem during porcine in vitro fertilization (IVF). After in vitro culture of porcine zygotes, only a low percentage of blastocysts develop and their quality is inferior to that of in vivo derived blastocysts. It is unknown whether the cytoplasmic maturation of the oocyte is sufficiently sustained in current in vitro maturation (IVM) procedures. The complex interplay between oocyte and cumulus cells during IVM is a key factor in this process. By focusing on this bidirectional communication, it is possible to control the coordination of cumulus expansion, and nuclear and cytoplasmic maturation during IVM to some extent. Therefore, this review focuses on the regulatory mechanisms between oocytes and cumulus cells to further the development of new in vitro embryo production (IVP) procedures, resulting in less polyspermy and improved oocyte developmental potential. Specifically, we focused on the involvement of cAMP in maturation regulation and function of oocyte-secreted factors (OSFs) in the bidirectional regulatory loop between oocyte and cumulus cells. Our studies suggest that maintaining high cAMP levels in the oocyte during the first half of IVM sustained improved oocyte maturation, resulting in an enhanced response after IVF and cumulus matrix disassembly. Recent research indicated that the addition of OSFs during IVM enhanced the developmental competence of small follicle-derived oocytes, which was stimulated by epidermal growth factor (EGF) via developing EGF-receptor signaling

Dynamics of 5-methylcytosine and 5-hydroxymethylcytosine during pronuclear development in equine zygotes produced by ICSI

Background: Global epigenetic reprogramming is considered to be essential during embryo development to establish totipotency. In the classic model first described in the mouse, the genome-wide DNA demethylation is asymmetric between the paternal and the maternal genome. The paternal genome undergoes ten-eleven translocation (TET)-mediated active DNA demethylation, which is completed before the end of the first cell cycle. Since TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine, the latter is postulated to be an intermediate stage toward DNA demethylation. The maternal genome, on the other hand, is protected from active demethylation and undergoes replication-dependent DNA demethylation. However, several species do not show the asymmetric DNA demethylation process described in this classic model, since 5-methylcytosine and 5-hydroxymethylcytosine are present during the first cell cycle in both parental genomes. In this study, global changes in the levels of 5-methylcytosine and 5-hydroxymethylcytosine throughout pronuclear development in equine zygotes produced in vitro were assessed using immunofluorescent staining. Results: We were able to show that 5-methylcytosine and 5-hydroxymethylcytosine both were explicitly present throughout pronuclear development, with similar intensity levels in both parental genomes, in equine zygotes produced by ICSI. The localization patterns of 5-methylcytosine and 5-hydroxymethylcytosine, however, were different, with 5-hydroxymethylcytosine homogeneously distributed in the DNA, while 5-methylcytosine tended to be clustered in certain regions. Fluorescence quantification showed increased 5-methylcytosine levels in the maternal genome from PN1 to PN2, while no differences were found in PN3 and PN4. No differences were observed in the paternal genome. Normalized levels of 5-hydroxymethylcytosine were preserved throughout all pronuclear stages in both parental genomes. Conclusions: In conclusion, the horse does not seem to follow the classic model of asymmetric demethylation as no evidence of global DNA demethylation of the paternal pronucleus during the first cell cycle was demonstrated. Instead, both parental genomes displayed sustained and similar levels of methylation and hydroxymethylation throughout pronuclear development