Oxidative Stress in Mouse Sperm Impairs Embryo Development, Fetal Growth and Alters Adiposity and Glucose Regulation in Female Offspring

<div><p>Paternal health cues are able to program the health of the next generation however the mechanism for this transmission is unknown. Reactive oxygen species (ROS) are increased in many paternal pathologies, some of which program offspring health, and are known to induce DNA damage and alter the methylation pattern of chromatin. We therefore investigated whether a chemically induced increase of ROS in sperm impairs embryo, pregnancy and offspring health. Mouse sperm was exposed to 1500 µM of hydrogen peroxide (H₂O₂), which induced oxidative damage, however did not affect sperm motility or the ability to bind and fertilize an oocyte. Sperm treated with H₂O₂ delayed on-time development of subsequent embryos, decreased the ratio of inner cell mass cells (ICM) in the resulting blastocyst and reduced implantation rates. Crown-rump length at day 18 of gestation was also reduced in offspring produced by H₂O₂ treated sperm. Female offspring from H₂O₂ treated sperm were smaller, became glucose intolerant and accumulated increased levels of adipose tissue compared to control female offspring. Interestingly male offspring phenotype was less severe with increases in fat depots only seen at 4 weeks of age, which was restored to that of control offspring later in life, demonstrating sex-specific impacts on offspring. This study implicates elevated sperm ROS concentrations, which are common to many paternal health pathologies, as a mediator of programming offspring for metabolic syndrome and obesity.</p></div

Implantation and placental and fetal parameters at day 18 of gestation.

<p>Values are mean ± SEM. n = 48 blastocyst stage embryos/treatment produced from 4 stud CBAF1 males transferred to contralateral uteri of 8 recipient females.</p><p>*indicates significantly different from control (also indicated by bold text) (P<0.05).</p>+<p>Indicates trending different from control P = 0.06. Survival rates post warming was between 90–95% for all treatment groups and did not differ statistically between treatment groups.</p

Effect of treatment of sperm with 1500 µM H₂O₂ on female offspring weight gain and glucose tolerance.

<p>(A) pre-weaning weight, (B) post weaning weight, (C) glucose tolerance (AUC) Control female (n = 7 from 6 litters), H₂O₂ female (n = 9 from 5 litters) representative of 15 stud CBAF1 males. Values are mean ± SEM.^* Indicates significantly different from control (P<0.05).</p

Post mortem male offspring body composition and fasting blood metabolites sired by control sperm or sperm treated with 1500 µM H₂O₂ at 17 weeks of age.

<p>Values represent mean ± SEM Control n = 14 males from 6 litters, H₂O₂ n = 7 males from 5 litters representative of 15 stud CBAF1 males.</p><p>*Indicates significantly different from control male offspring (also indicated by bold text) (P<0.05).</p

Effect of treating sperm with 1500 µM H₂O₂ on subsequent (A) blastocyst cell number, (B) trophectoderm cell number, (C) inner cell mass (ICM) cell number, (D) percentage (%) of ICM/total cell number.

<p>Control (n = 84 embryos), H₂O₂ (n = 79 embryos). Values are mean ± SEM.^* Indicates significantly different from control (P<0.05) ^** indicates significantly different from control (P<0.01).</p

Slow freezing and vitrification of mouse morula and early blastocysts

PurposeTo assess the relative success of morula and early blastocyst slow freezing and vitrification in regards to survival and implantation rates utilising protocols which could be clinically implemented as a viable alternative to expanded blastocyst stage freezing.MethodsMouse morula and early blastocysts were either slow frozen/thawed or vitrified/warmed. Their subsequent survival, blastocyst development and blastocyst cell number and allocation to either the inner cell mass, trophectoderm or epiblast was assessed. In addition blastocysts were also transferred to pseudopregnant recipients and implantation and fetal development was determined.ResultsVitrification of both morula and early blastocysts resulted in significantly higher rates of survival and blastocyst development compared to slow freezing. In addition slow frozen early blastocysts had significantly reduced blastocyst cell number compared to control however vitrified morula and early blasocyts and slow frozen morula had equivocal blastocyst cell numbers. Transfer of blastocysts from both methods of cryopreservation resulted in similar implantation rates however the placentas created from slow frozen early blastocysts were significantly lighter than control (95.5 g ± 5.4 vs. 122.0 g ± 4.2 respectively).ConclusionsVitrification resulted in significantly higher rates of morula and early blastocyst survival and blastocyst development compared to slow freezing. In addition this study has validated the use of a closed DMSO free vitrification protocol which could then be investigated for use in the clinical setting as an alternative to expanded blastocyst freezing.Deirdre Zander-Fox, Michelle Lane, Hamish Hamilto

Mitochondrial SIRT5 is present in follicular cells and is altered by reduced ovarian reserve and advanced maternal age

Women with reduced ovarian reserve or advanced maternal age have an altered metabolic follicular microenvironment. As sirtuin 5 (SIRT5) senses cellular metabolic state and post-translationally alters protein function, its activity may directly impact on oocyte viability and pregnancy outcome. Therefore, we investigated the role of SIRT5 in relation to ovarian reserve and maternal age. Women (n=47) undergoing routine IVF treatment were recruited and allocated to one of three cohorts based on ovarian reserve and maternal age. Surplus follicular fluid, granulosa and cumulus cells were collected. SIRT5 mRNA, protein and protein activity was confirmed in granulosa and cumulus cells via qPCR, immunohistochemistry, western blotting and desuccinylation activity. The presence of carbamoyl phosphate synthase I (CPS1), a target of SIRT5, was investigated by immunohistochemistry and follicular-fluid ammonium concentrations determined via microfluorometry. Women with reduced ovarian reserve or advanced maternal age had decreased SIRT5 mRNA, protein and desuccinylation activity in granulosa and cumulus cells resulting in an accumulation of follicular-fluid ammonium, presumably via alterations in activity of a SIRT5 target, CPS1, which was present in granulosa and cumulus cells. This suggests a role for SIRT5 in influencing oocyte quality and IVF outcomes.Leanne Pacella-Ince, Deirdre L. Zander-Fox and Michelle Lan