CXCL5-CXCR2 signaling is a senescence-associated secretory phenotype in preimplantation embryos.

Pregnancy rate of women decreases with age due to declining quality of oocytes and embryos. However, there is no established method to improve pregnancy rate in aging women. In this study, we identified a senescence-associated secretory phenotype (SASP) factor partially responsible for the decline in embryo implantation potential. Based on microarray analysis using young and aging human embryos at the same morphological grade, 702 genes showed >fivefold increases in aging human blastocysts. Among these genes, C-X-C motif chemokine 5 (CXCL5) showed 7.7-fold increases in aging human blastocysts. However, no-age-dependent changes in expression of the CXCR2, the cognate receptor for CXCL5, were found. In aging mice, Cxcl5 transcript levels were also increased in oocytes and embryos. Treatment of young mouse embryos with CXCL5 decreased implantation rates, together with increased expression of aging markers (P53, P21, Pai-1, and Il-6). Moreover, CXCL5 treatment suppressed trophoblast outgrowth in young mouse blastocysts. Conversely, suppression of CXCL5-CXCR2 signaling in aging mouse embryos using neutralizing antibodies and a receptor antagonist improved the implantation rate, leading to increases in pregnancy and delivery of normal pups. The gene expression pattern of these embryos was comparable to that in young mouse embryos showing enriched cell proliferation-related pathways. In conclusion, we identified CXCL5 as a SASP factor in human and mouse embryos and suppression of CXCL5-CXCR2 signaling during embryo culture improved pregnancy success in aging mice. Future analysis on CXCL5-CXCR2 signaling suppression in human embryos could be the basis to improve embryo development and pregnancy outcome in middle-aged infertile patients.

Erratum: J Reprod Dev, Vol. 64, No. 2, p. 153-160, Macromolecular crowded conditions strengthen contacts between mouse oocytes and companion granulosa cells during in vitro growth.

Figure 7 have been corrected.

Macromolecular crowded conditions strengthen contacts between mouse oocytes and companion granulosa cells during in vitro growth.

Macromolecular crowded culture medium formed by addition of polyvinylpyrrolidone (PVP; molecular weight = 360 000), positively influences the viability, growth, and development of bovine oocytes. Owing to its apparently various effects, uncovering the specific mechanisms of crowding responsible for these outcomes is important. The present study was conducted to determine the effects of crowding on oocytes with a particular focus on the intimacy of contacts between oocyte and cumulus/granulosa cells. Growing mouse oocyte-granulosa cell complexes were cultured for 10 days in a modified α-minimum essential medium, supplemented with PVP at a concentration of 0%, 1%, 2%, or 3% (w/v). Although the complexes developed in all groups, 2% and 3% PVP medium induced a substantial morphological modification, and a larger proportion of oocytes associated with cumulus cells survived in 3% PVP medium than in the 0% or 1% PVP medium. No significant difference was found in the frequencies of polar body extrusion (78-88%) and blastocyst formation (approximately 40%) after in vitro fertilization among the experimental groups. Confocal laser scanning microscopy indicated a higher number of transzonal processes reaching the oocyte from cumulus cells in 2% PVP medium than in 0% PVP medium. Transmission electron microscopy depicted close adhesion of the oocyte with cumulus cells in 2% PVP medium -bearing a resemblance to their in vivo counterparts- and loose adhesion in 0% PVP medium. In conclusion, we found that a mechanism for the action of crowded conditions involves the strengthening of contacts and communication between oocytes and companion cumulus/granulosa cells.

Mycoplasma ovis detected in free-living Japanese serows, Capricornis crispus.

Nineteen blood samples collected from free-ranging wild Japanese serows, Capricornis crispus, between 2006 and 2008 in Iwate prefecture were examined for the hemoplasma infection by real-time PCR targeting the 16S rRNA gene. Five (26.3%) out of the 19 samples were positive in real-time PCR with an average melting temperature at 85.18 °C. The positive samples in the real-time PCR were reconfirmed by conventional PCR, and one of them was successful for direct DNA sequencing. The nucleotide sequence of the 16S rRNA gene of the representative stain was identical to that of Mycoplasma ovis. This was the first demonstration of hemotropic mycoplasma infections among the free-living Japanese serows in Japan.

Novel hemoplasma species detected in free-ranging sika deer (Cervus nippon).

Hemoplasma infections in wild ungulates have not been reported yet in Japan. We examined presence of hemoplasmas in blood samples collected from 147 sika deer (Cervus nippon) in the Iwate prefecture by real-time PCR, and found 13 (9%) were positive. Almost entire region of the 16S rRNA gene of the representative strains from positive samples was amplified by conventional PCR. The nucleotide sequences of the 16S rRNA gene were further determined and compared with those of other hemoplasmas. Our examinations 1st revealed the presence of 2 distinct hemoplasma species in sika deer, which are previously not described. One of them was closely related to M. ovis by the 16S rRNA sequence analysis, but was found distinct by comparison of the RNase P RNA gene sequences. Pathogenicity of these two hemoplasma species in sika deer is currently unknown.

Phylogenetic analysis of the 16S rRNA gene of Anaplasma species detected from Japanese serows (Capricornis crispus).

Nineteen blood samples collected from free-living Japanese serows, Capricornis crispus, between 2006 and 2008 in Iwate prefecture were examined for the anaplasma infection by PCR amplification of a part of the 16S rRNA gene. Ten (52.6%) out of the 19 samples produced a visible band in electrophoresed agarose gels. Positive PCR products were subjected to DNA sequencing. We found the nucleotide sequences were identical. Almost entire length of the 16S rRNA gene for a representative stain was then sequenced. We found the nucleotide sequence of the 16S rRNA gene distinct from previously established Anaplasma species in phylogenetic analysis. Our data first indicated that anaplasma infection occurred continuously among the free-living Japanese serow populations in northern Japan.

Evidence for pestivirus infection in free-living Japanese serows, Capricornis crispus.

Sixteen serum samples collected from free-living Japanese serows, Capricornis crispus, between 2001 and 2004 in Morioka and its vicinity were examined for the presence of pestivirus by reverse transcription-nested PCR procedure. Three out of the 16 samples produced a visible band in electrophoresed agarose gels. The nucleotide sequences of the three PCR products were found to be identical. The pestivirus found in the serow was identified as Bovine viral diarrhea virus 1 (BVDV-1) based on nucleotide sequence analyses by phylogeny as well as palindromic nucleotide substitutions at the 5' untranslated regions. Our data first indicated that BVDV-1 infection occurred continuously among the free-living serow populations though the role of BVDV-1 in wild ungulates is currently unknown.