Advanced maternal age induces fetal growth restriction through decreased placental inflammatory cytokine expression and immune cell accumulation in mice

Advanced maternal age is a risk factor for female infertility, and placental dysfunction is considered one of the causes of pregnancy complications. We investigated the effects of advanced maternal aging on pregnancy outcomes and placental senescence. Female pregnant mice were separated into three groups: young (3 months old), middle (8–9 months old), and aged (11–13 months old). Although the body weights of young and middle dams gradually increased during pregnancy, the body weight of aged dams only increased slightly. The placental weight and resorption rate were significantly higher, and live fetal weights were reduced in a maternal age-dependent manner. Although mRNA expression of senescence regulatory factors (p16 and p21) increased in the spleen of aged dams, mRNA expression of p16 did not change and that of p21 was reduced in the placenta of aged dams. Using a cytokine array of proteins extracted from placental tissues, the expression of various types of senescence-associated secretory phenotype (SASP) factors was decreased in aged dams compared with young and middle dams. The aged maternal placenta showed reduced immune cell accumulation compared with the young placenta. Our present results suggest that models using pregnant mice older than 8 months are more suitable for verifying older human pregnancies. These findings suggest that general cellular senescence programs may not be included in the placenta and that placental functions, including SASP production and immune cell accumulation, gradually decrease in a maternal age-dependent manner, resulting in a higher rate of pregnancy complications.     

Upregulation of interferon-stimulated genes and interleukin-10 in peripheral blood immune cells during early pregnancy in dairy cows

In cows, interferon-tau (IFNT) regulates maternal recognition around days 15-19 after artificial insemination (AI). The present study hypothesized that if key target genes of IFNT are clearly upregulated in earlier stages of pregnancy, these genes could be use as indices of future pregnancy in cows. Therefore, we determined the expression of these genes in peripheral blood mononuclear leukocytes (PBMCs) and polymorphonuclear granulocytes (PMNs) during the maternal recognition period (MRP). Twenty multiparous Holstein cows were subjected to AI on day 0 and categorized into the following groups: pregnancy (Preg, n = 9), embryonic death (ED, n = 5) and non-pregnancy (NP, n = 6). Progesterone levels in the Preg group were higher than those in the NP group on days 12-21. ISG15 and OAS-1 (IFN-stimulated genes: ISGs) mRNA in PBMCs on day 8 was higher in the Preg group than in the NP group, and these mRNAs in PMNs was higher in the Preg group on day 5 than in the NP and ED groups. Interleukin-10 (IL-10, Th2 cytokine) mRNA expression increased on day 8 in the PBMCs of pregnant cows. Tumor necrosis factor α (TNFα, Th1 cytokine) mRNA expression was stable in all groups. In an in vitro cell culture experiment, IFNT stimulated mRNA expression of ISGs in both PBMCs and PMNs. IFNT stimulated IL-10 mRNA expression in PBMCs, whereas IFNT increased TNFα mRNA levels in PBMCs in vitro. The results suggest that ISGs and IL-10 could be responsive to IFNT before the MRP in peripheral blood immune cells and may be useful target genes for reliable indices of pregnancy before the MRP.     

Expression of mRNA for cell adhesion molecules in the bovine corpus luteum during the estrous cycle and PGF2alpha-induced luteolysis

Cell-to-cell interaction via cell contact-dependent pathway is essentially important for maintenance and regulation of corpus luteum (CL) integrity and its physiological actions. The objective of the present study was to evaluate the mRNA expression of the cell adhesion molecules (CAMs) that are constituent factors of gap junctions [connexin (Cx) 43] and adherence junctions (VE-, E-, N-cadherin) in two types of endothelial cells from the mid CL and in CL tissue during the estrous cycle and PGF(2alpha)-induced luteolysis in the cow. Specific mRNA expression for Cx43 and N-cadherin was detected in cytokeratin-positive (CK+) and cytokeratin-negative (CK-) luteal endothelial cells (EC) and fully luteinized granulosa cells (LGC). E-cadherin mRNA was expressed in CK+EC and LGC, but not in CK-EC. VE-cadherin mRNA was expressed in both CK+ and CK-EC. During the estrous cycle, Cx43 mRNA expression was significantly lower in the regressing CL. VE-cadherin expression also tended to increase in the mid CL and increased significantly in the regressing CL. E-cadherin mRNA expression was higher in the early and late CL than in the mid- and regressing CL. N-cadherin mRNA expression gradually increased from the early to late CL followed by a decrease in the regressing CL. During PGF(2alpha)-induced luteolysis, Cx43 mRNA expression appeared to increase, and VE-cadherin and E-cadherin mRNA significantly increased at 24 h. N-cadherin mRNA expression decreased 2 and 4 h after PGF(2alpha) administration. Collectively, expression of the mRNAs for CAMs was different in the two types of luteal endothelial cells and fully luteinized granulosa cells and changed independently in the CL during the estrous cycle and PGF(2alpha)-induced luteolysis in the cow. The results suggest that CAMs play physiological roles in cell-to-cell communication to regulate both gap and adherence junctions during CL development and regression in the cow.     

Effects of reaggregated granulosa cells and oocytes derived from early antral follicles on the properties of oocytes grown in vitro

In this study, we examined the effects of reconstructed oocyte–granulosa cell complexes (OGCs) on the development of porcine oocytes derived from early antral follicles (EAFs; 0.5–0.7 mm in diameter). When denuded oocytes were cocultured with granulosa cells derived from other EAFs, the oocytes and granulosa cells aggregated to form OGCs after 2 days of culture. After 14 days of culture, we compared cell number, oocyte diameter, and oocyte chromatin configuration in unmanipulated (natural) OGCs, reconstructed OGCs, and OGCs collected from antral follicles (AFs, 3.0–6.0 mm in diameter). The diameters of oocytes from reconstructed OGCs grown in vitro were not different from those of oocytes from natural OGCs, although they were significantly smaller than those of oocytes from antral follicle (AF) OGCs. Oocyte chromatin configuration did not differ among the 3 OGC groups, but the oocyte nuclear maturation rate was lower in the reconstructed OGCs and higher in the AF OGCs. However, when the in vitro culture period for the reconstructed OGCs was extended by 2 days, the nuclear maturation rate of oocytes from reconstructed OGCs was similar to that of oocytes from natural OGCs. In addition, blastocysts were successfully obtained from oocytes from reconstructed OGCs. In conclusion, we established an innovative culture method that allows oocytes and granulosa cells from EAFs to reaggregate as reconstructed OGCs, which yield oocytes with the ability to develop to the blastocyst stage.     

The Effect of Single Nucleotide Polymorphisms in the Tumor Necrosis Factor-α Gene on Reproductive Performance and Immune Function in Dairy Cattle

The present study aimed to assess the effect of polymorphisms in the tumor necrosis factor α (TNF-α) promoter (A/A, A/G and G/G) and exons (T/T, T/C and C/C) on immune function and reproductive performance in dairy cows. The occurrence of the first postpartum ovulation within 3 weeks in the cows with the TNF-α promoter A/G and G/G genotypes was higher than in the A/A group. Among the different TNF-α exon genotypes, the occurrence of early first postpartum ovulation was higher in the T/C and C/C genotype groups than in the T/T group. Single nucleotide polymorphisms (SNPs) in the TNF-α gene did not affect the rate of artificial insemination (AI) or duration from parturition to next conception (days open). The apoptosis rate of polymorphonuclear leukocytes (PMNs) did not differ among the TNF-α promoter genotypes, but the PMN transmigration rate was significantly higher for the A/A and A/G genotypes than for the G/G genotype. Interleukin 8 (IL-8) mRNA expression in PMNs and peripheral blood mononuclear cells (PBMCs) before culture was significantly higher for the A/A genotype compared with the G/G genotype. There were no significant differences between the genotypes in the mRNA expression of TNF-α, IL-6, IL-1β, and toll-like receptor 4 (TLR4) in PMNs and PBMCs before and 4 h after culture. IL-8 and IL-1β production by PBMCs cultured for 4 h was significantly higher for the animals with the A/A genotype than for those with the G/G genotype. On the other hand, no significant difference was observed in IL-8 and IL-1β production by PMNs among different TNF-α genotypes. Taken together, these results suggest that SNP in the TNF-α gene affects immune function and reproductive performance in dairy cows.     

Effect of intraluteal injection of endothelin type A receptor antagonist on PGF2alpha-induced luteolysis in the cow

Endothelin-1 (ET-1) is a luteolytic mediator in the bovine corpus luteum (CL), and its action appears to be via endothelin type A receptor (ETR-A). Thus, the aim of the present study was to determine the effect of ETR-A antagonist on PGF2alpha-induced luteolysis in the cow. Cows on days 10-12 of the estrous cycle were subjected to five intraluteal injections of the ETR-A antagonist LU 135252 in saline or only saline at -0.5, 2, 4, 6, and 8 h after PGF2alpha administration (=0 h). Serial luteal biopsies were conducted to determine the expression of mRNA in the luteal tissue. There were no significant differences in the decrease in plasma progesterone (P) concentrations and the mRNA expressions of steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase/Delta5, Delta4-isomerase between the ETR-A antagonist-treated group and the control group. However, the start of the decline in CL volume and blood flow area surrounding the CL was delayed for almost two days in the ETR-A antagonist-treated group compared to the control group. The mRNA expression of preproET-1 and endothelin type B receptor increased in both groups, while the ETR-A mRNA remained unchanged. In addition, caspase-3 mRNA expression increased significantly at 24 h in the control group only and its level was higher than that of the ETR-A antagonist-treated group. Thus, the present study suggests that ET-1 regulates structural luteolysis via ETR-A by controlling blood vessel contraction in the CL of the cow.     

Relationships between the first ovulation postpartum and polymorphism in genes relating to function of immunity, metabolism and reproduction in high-producing dairy cows

The decrease in fertility and conception rates of high-producing dairy cows is one of the major negative impacts for today's producers. The recovery of ovarian activity postpartum is affected by the status of immunity, metabolism and reproduction and plays a critical role in subsequent fertility after parturition in the cow. In the present study we investigated the relationships between polymorphisms in genes relating to the above functions and the first postpartum ovulation as a marker of the recovery of ovarian function in the cow. In immune function related-factors, the occurrence of first postpartum ovulation within 3 weeks in the C/C genotypes of tumor necrosis factor α (TNFα) exon (55.4%) and the A/G genotypes of TNFα promoter (55.4%) was significantly higher than that in T/T genotypes of TNFα exon (14.3%) and A/A genotypes of TNFα promoter (14.3%). Moreover, anovulatory cows with the T/T genotype of TNFα exon and the A/A genotype of TNFα promoter tended to have a prolonged days open compared with those of the other genotypes of TNFα polymorphisms. In metabolic function-related factors, ovulatory and anovulatory cows had a different distribution for alleles of the growth hormone receptor, but there were no significant differences in genotype and allele frequency of insulin-like growth factor-I polymorphism. No significant relationships were found between ovarian function after parturition and polymorphisms for reproduction-related genes. In conclusion, polymorphisms of TNFα gene both in exon and promoter regions have a strong association with the early first ovulation within 3 weeks after parturition in the high-producing dairy cow. Taken together, polymorphisms of TNFα gene could be strongly related to early first ovulation after parturition, thus being an effective tool of selection for improving reproductive performance in the high-producing dairy cow.