PolyADP-Ribosylation Is Required for Pronuclear Fusion during Postfertilization in Mice

Background

During fertilization, pronuclear envelope breakdown (PNEB) is followed by the mingling of male and female genomes. Dynamic chromatin and protein rearrangements require posttranslational modification (PTM) for the postfertilization development.

Methodology/Principal Findings

Inhibition of poly(ADP-ribose) polymerase activity (PARylation) by either PJ-34 or 5-AIQ resulted in developmental arrest of fertilized embryos at the PNEB. PARylation inhibition affects spindle bundle formation and phosphorylation of Erk molecules of metaphase II (MII) unfertilized oocytes. We found a frequent appearance of multiple pronuclei (PN) in the PARylation-inhibited embryos, suggesting defective polymerization of tubulins. Attenuated phosphorylation of lamin A/C by PARylation was detected in the PARylation-inhibited embryos at PNEB. This was associated with sustained localization of heterodomain protein 1 (HP1) at the PN of the one-cell embryos arrested by PARylation inhibition.

Conclusions/Significance

Our findings indicate that PARylation is required for pronuclear fusion during postfertilization processes. These data further suggest that PARylation regulates protein dynamics essential for the beginning of mouse zygotic development. PARylation and its involving signal-pathways may represent potential targets as contraceptives.     

Expression analysis of the NLRP gene family suggests a role in human preimplantation development

Background

The NLRP (Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing) family, also referred to as NALP family, is well known for its roles in apoptosis and inflammation. Several NLRPs have been indicated as being involved in reproduction as well.

Methodology

We studied, using the unique human gametes and embryo materials, the expression of the NLRP family in human gametes and preimplantation embryos at different developmental stages, and compared the expression levels between normal and abnormal embryos using real-time PCR.

Principal Findings

Among 14 members of the NLRP family, twelve were detected in human oocytes and preimplantation embryos, whereas seven were detected in spermatozoa. Eight NLRPs (NLRP4, 5, 8, 9, 11, 12, 13, and 14) showed a similar expression pattern: their expression levels were high in oocytes and then decreased progressively in embryos, resulting in a very low level in day 5 embryos. However, NLRP2 and NLRP7 showed a different expression pattern: their expression decreased from oocytes to the lowest level by day 3, but increased again by day 5. The expression levels of NLRP5, 9, and 12 were lower in day 1 abnormal embryos but higher in day3 and day5 arrested embryos, when compared with normal embryos at the same stages. NLRP7 was down-regulated in day 1 and day 5 abnormal embryos but over-expressed in day3 arrested embryos.

Conclusions

According to our results, different NLRPs possibly work in a stage-dependent manner during human preimplantation development.     

Does the Number of Oocytes Retrieved Influence Pregnancy after Fresh Embryo Transfer?

Background

The nature of the association between the number of oocytes retrieved and in vitro fertilization (IVF) outcomes after fresh embryo transfer remains unclear because of conflicting results reported in the studies on this subject. In addition, the influence of the quality of the embryos transferred is usually neglected. The objective of this study is to assess the relationships of the number of oocytes retrieved, the number and quality of embryos transferred, and the prospects of pregnancy after fresh embryo transfer.

Methods

The data on 3131 infertile women undergoing their first IVF treatment cycle between January 2009 and December 2010 were collected retrospectively. Restricted cubic splines and stratified analyses were used to explore the relationships between the number of oocytes retrieved, the number and quality of embryos transferred, and the IVF outcomes.

Results

When stratified by the number and quality of transferred embryos, no significant differences in the chances for clinical pregnancy and live birth were found in three groups of oocytes yielded (≤6, 7–14, or ≥15). The relationship between the number of oocytes retrieved and pregnancy is nearly a reflection of the pattern of the relationship between the number of oocytes retrieved and the probability of having two good-quality embryos transferred. The patients with the “optimal” number of oocytes were not only younger but also had the highest probability of having two good-quality embryos replaced.

Conclusions

Similarly aged patients have similar pregnancy prospects after fresh embryo transfer when the same number and quality of embryos are replaced, irrespective of their number of oocytes. Selecting the desired number of good-quality embryos for transfer is the key to IVF success. Thus, aiming at retrieving an optimal number of oocytes to maximize IVF outcomes in a fresh cycle could place undue stress on the patients and may not be the best medical decision.     

Polyembryony in non-apomictic citrus genotypes

Background and Aims

Adventitious embryony from nucellar cells is the mechanism leading to apomixis in Citrus sp. However, singular cases of polyembryony have been reported in non-apomictic genotypes as a consequence of 2x × 4x hybridizations and in vitro culture of isolated nucelli. The origin of the plants arising from the aforementioned processes remains unclear.

Methods

The genetic structure (ploidy and allelic constitution with microsatellite markers) of plants obtained from polyembryonic seeds arising from 2x × 4x sexual hybridizations and those regenerated from nucellus culture in vitro was systematically analysed in different non-apomictic citrus genotypes. Histological studies were also conducted to try to identify the initiation process underlying polyembryony.

Key Results

All plants obtained from the same undeveloped seed in 2x × 4x hybridizations resulted from cleavage of the original zygotic embryo. Also, the plants obtained from in vitro nucellus culture were recovered by somatic embryogenesis from cells that shared the same genotype as the zygotic embryos of the same seed.

Conclusions

It appears that in non-apomictic citrus genotypes, proembryos or embryogenic cells are formed by cleavage of the zygotic embryos and that the development of these adventitious embryos, normally hampered, can take place in vivo or in vitro as a result of two different mechanisms that prevent the dominance of the initial zygotic embryo.     

Release of sICAM-1 in Oocytes and In Vitro Fertilized Human Embryos

Background

During the last years, several studies have reported the significant relationship between the production of soluble HLA-G molecules (sHLA-G) by 48–72 hours early embryos and an increased implantation rate in IVF protocols. As consequence, the detection of HLA-G modulation was suggested as a marker to identify the best embryos to be transferred. On the opposite, no suitable markers are available for the oocyte selection.

Methodology/Principal Findings

The major finding of the present paper is that the release of ICAM-1 might be predictive of oocyte maturation. The results obtained are confirmed using three independent methodologies, such as ELISA, Bio-Plex assay and Western blotting. The sICAM-1 release is very high in immature oocytes, decrease in mature oocytes and become even lower in in vitro fertilized embryos. No significant differences were observed in the levels of sICAM-1 release between immature oocytes with different morphological characteristics. On the contrary, when the mature oocytes were subdivided accordingly to morphological criteria, the mean sICAM-I levels in grade 1 oocytes were significantly decreased when compared to grade 2 and 3 oocytes.

Conclusions/Significance

The reduction of the number of fertilized oocytes and transferred embryos represents the main target of assisted reproductive medicine. We propose sICAM-1 as a biochemical marker for oocyte maturation and grading, with a possible interesting rebound in assisted reproduction techniques.     

Chronic Unpredictable Stress Decreases Expression of Brain-Derived Neurotrophic Factor (BDNF) in Mouse Ovaries: Relationship to Oocytes Developmental Potential

Background

Brain-derived neurotropic factor (BDNF) was originally described in the nervous system but has been shown to be expressed in ovary tissues recently, acting as a paracrine/autocrine regulator required for developments of follicles and oocytes. Although it is generally accepted that chronic stress impairs female reproduction and decreases the expression of BDNF in limbic structures of central nervous system, which contributes to mood disorder. However, it is not known whether chronic stress affects oocytes developments, nor whether it affects expression of BDNF in ovary.

Methods

Mice were randomly assigned into control group, stressed group, BDNF-treated group and BDNF-treated stressed group. The chronic unpredictable mild stress model was used to produce psychosocial stress in mice, and the model was verified by open field test and hypothalamic-pituitary-adrenal (HPA) axis activity. The methods of immunohistochemistry and western blotting were used to detect BDNF protein level and distribution. The number of retrieved oocytes, oocyte maturation, embryo cleavage and the rates of blastocyst formation after parthenogenetic activation were evaluated.

Results

Chronic unpredictable stress decreased the BDNF expression in antral follicles, but didn’t affect the BDNF expression in primordial, primary and secondary follicles. Chronic unpredictable stress also decreased the number of retrieved oocytes and the rate of blastocyst formation, which was rescued by exogenous BDNF treatment.

Conclusion

BDNF in mouse ovaries may be related to the decreased number of retrieved oocytes and impaired oocytes developmental potential induced by chronic unpredictable stress.     

Effects of thioglycolic acid on parthenogenetic activation of Xenopus oocytes

Background

Existing in Permanent-wave solutions (PWS), thioglycolic acid (TGA) is widely used in hairdressing industry for its contribution to hair styling. However, the toxicity of TGA, especially its reproductive toxicity, gradually calls the attention of more and more researchers.

Method

In this work, xenopus oocytes were pretreated with different concentration of TGA, and then activated by calcium ionophore A23187. During culture, the oocytes activation rates were taken note at different time after adding calcium ionophore A23187. At the end of the culture period, the nuclear status was detected under confocal microscope. In addition, some other samples were collected for Western-Blotting analysis.

Result

TGA significantly inhibited the oocytes activation rate and pronuclear formation. It may be resulted from the inhibition of the degradation of p-ERK1, Mos and CyclinB2.

Conclusion

TGA inhibits in vitro parthenogenetic activation of xenopus oocytes with inhibited the degradation of proteins involved in mitogenic-activated protein kinase (MAPK) and maturation-promoting factor (MPF) pathways.     

Polyamines, IAA and ABA during germination in two recalcitrant seeds: Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm)

Background and Aims

Plant growth regulators play an important role in seed germination. However, much of the current knowledge about their function during seed germination was obtained using orthodox seeds as model systems, and there is a paucity of information about the role of plant growth regulators during germination of recalcitrant seeds. In the present work, two endangered woody species with recalcitrant seeds, Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm), native to the Atlantic Rain Forest, Brazil, were used to study the mobilization of polyamines (PAs), indole-acetic acid (IAA) and abscisic acid (ABA) during seed germination.

Methods

Data were sampled from embryos of O. odorifera and embryos and megagametophytes of A. angustifolia throughout the germination process. Biochemical analyses were carried out in HPLC.

Key Results

During seed germination, an increase in the (Spd + Spm) : Put ratio was recorded in embryos in both species. An increase in IAA and PA levels was also observed during seed germination in both embryos, while ABA levels showed a decrease in O. odorifera and an increase in A. angustifolia embryos throughout the period studied.

Conclusions

The (Spd + Spm) : Put ratio could be used as a marker for germination completion. The increase in IAA levels, prior to germination, could be associated with variations in PA content. The ABA mobilization observed in the embryos could represent a greater resistance to this hormone in recalcitrant seeds, in comparison to orthodox seeds, opening a new perspective for studies on the effects of this regulator in recalcitrant seeds. The gymnosperm seed, though without a connective tissue between megagametophyte and embryo, seems to be able to maintain communication between the tissues, based on the likely transport of plant growth regulators.     

Parthenogenic Blastocysts Derived from Cumulus-Free In Vitro Matured Human Oocytes

Background

Approximately 20% of oocytes are classified as immature and discarded following intracytoplasmic sperm injection (ICSI) procedures. These oocytes are obtained from gonadotropin-stimulated patients, and are routinely removed from the cumulus cells which normally would mature the oocytes. Given the ready access to these human oocytes, they represent a potential resource for both clinical and basic science application. However culture conditions for the maturation of cumulus-free oocytes have not been optimized. We aimed to improve maturation conditions for cumulus-free oocytes via culture with ovarian paracrine/autocrine factors identified by single cell analysis.

Methodology/Principal Finding

Immature human oocytes were matured in vitro via supplementation with ovarian paracrine/autocrine factors that were selected based on expression of ligands in the cumulus cells and their corresponding receptors in oocytes. Matured oocytes were artificially activated to assess developmental competence. Gene expression profiles of parthenotes were compared to IVF/ICSI embryos at morula and blastocyst stages. Following incubation in medium supplemented with ovarian factors (BDNF, IGF-I, estradiol, GDNF, FGF2 and leptin), a greater percentage of oocytes demonstrated nuclear maturation and subsequently, underwent parthenogenesis relative to control. Similarly, cytoplasmic maturation was also improved as indicated by development to blastocyst stage. Parthenogenic blastocysts exhibited mRNA expression profiles similar to those of blastocysts obtained after IVF/ICSI with the exception for MKLP2 and PEG1.

Conclusions/Significance

Human cumulus-free oocytes from hormone-stimulated cycles are capable of developing to blastocysts when cultured with ovarian factor supplementation. Our improved IVM culture conditions may be used for obtaining mature oocytes for clinical purposes and/or for derivation of embryonic stem cells following parthenogenesis or nuclear transfer.