Quantitative phosphoproteomics explain cryopreservation-induced reductions in ram sperm motility.

Sperm cryopreservation decreases motility, probably due to changes in protein phosphorylation. Our objective was to use quantitative phosphoproteomics for systematic comparative analyses of fresh versus frozen-thawed sperm to identify factors causing cryo-injury. Ejaculates were collected (artificial vagina) from six Dorper rams, pooled, extended, and frozen over liquid nitrogen. Overall, 915, 3382, and 6875 phosphorylated proteins, phosphorylated peptides, and phosphorylation sites, respectively, were identified. At least two modified sites were present in 57.94% of the 6875 phosphosites identified, of which AKAP4 protein contained up to 331 modified sites. There were 732 phosphorylated peptides significantly up-regulated and 909 significantly down-regulated in frozen-thawed versus fresh sperm. Moreover, the conserved motif [RxxS] was significantly down-regulated in frozen-thawed sperm. Phosphorylation of sperm-specific proteins, e.g., AKAP3/4, CABYR, FSIP2, GSK3A/B, GPI, and ODF1/2 make them potential biomarkers to assess the quality of frozen-thawed ram sperm. Furthermore, these differentially phosphorylated proteins and modification sites were implicated in cryopreservation-induced changes in sperm energy production, fiber sheath composition, and various biological processes. We concluded that abnormal protein phosphorylation modifications are key regulators of reduced sperm motility. These novel findings implicated specific protein phosphorylation modifications in sperm cryo-injury. SIGNIFICANCE: This study used phosphorylated TMT quantitative proteomics to explore regulation of epigenetic modifications in frozen-thawed ram sperm. This experiment demonstrated that ram sperm freezing affects phosphorylation site modifications of proteins, especially those related to functions such as sperm motility and energy production. Furthermore, it is important to link functions of phosphorylated proteins with changes in sperm quality after freezing and thawing, and to clarify intrinsic reasons for sperm quality changes, which is of great importance for elucidating mechanisms of sperm freezing damage. Based on these protein markers and combined with cryoprotectant design theory, it provides a theoretical basis and data reference to study sperm cryoprotectants.

Melatonin Promotes in vitro Development of Vitrified-Warmed Mouse GV Oocytes, Potentially by Modulating Phosphorylation of Drp1.

Previous studies have shown that melatonin can mitigate cryopreservation-induced mitochondrial dysfunction in oocytes; however, the underlying molecular mechanism remains unclear. The objective of the present study was to investigate whether melatonin can improve the mitochondrial function during in vitro maturation of vitrified-warmed mouse germinal vesicle (GV) oocytes by modulating phosphorylation of dynamin related protein 1 (Drp1). Vitrification/warming procedures resulted in the following: (1) After cryopreservation of mouse GV oocytes, the phosphorylation level of Drp1 at Ser616 (p-Drp1 Ser616) in metaphase II (MII) oocytes was increased (P < 0.05). Furthermore, the rates of in vitro maturation, cleavage and blastocyst formation after parthenogenetic activation were decreased (P < 0.05). (2) In MII oocytes, the expression levels of translocase of the mitochondrial outer membrane 20 (TOMM20), mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) content, and mRNA levels of mitochondrial biogenesis-related genes (Sirt1, Pgc-1α, Tfam) were all decreased (P < 0.05), and (3) Reactive oxygen species (ROS) level, early apoptosis level, Cytochrome C release and mRNA levels of pro-apoptotic related genes (Bax, Caspase9, Caspase3) in MII oocytes were all increased (P < 0.05). The results of this study further revealed that negative impacts of GV oocyte cryopreservation were mitigated by supplementation of warming and in vitro maturation media with 10-7mol /L melatonin or 2 x 10-5mol/L Mdivi-1 (Drp1 inhibitor). Therefore, we concluded that 10-7mol/L melatonin improved mitochondrial function, reduced oxidative stress and inhibited apoptosis by regulating phosphorylation of Drp1, thereby enhancing in vitro development of vitrified-warmed mouse GV oocytes.

Melatonin improves the first cleavage of parthenogenetic embryos from vitrified-warmed mouse oocytes potentially by promoting cell cycle progression.

BACKGROUND: This study investigated the effect of melatonin (MT) on cell cycle (G1/S/G2/M) of parthenogenetic zygotes developed from vitrified-warmed mouse metaphase II (MII) oocytes and elucidated the potential mechanism of MT action in the first cleavage of embryos. RESULTS: After vitrification and warming, oocytes were parthenogenetically activated (PA) and in vitro cultured (IVC). Then the spindle morphology and chromosome segregation in oocytes, the maternal mRNA levels of genes including Miss, Doc1r, Setd2 and Ythdf2 in activated oocytes, pronuclear formation, the S phase duration in zygotes, mitochondrial function at G1 phase, reactive oxygen species (ROS) level at S phase, DNA damage at G2 phase, early apoptosis in 2-cell embryos, cleavage and blastocyst formation rates were evaluated. The results indicated that the vitrification/warming procedures led to following perturbations 1) spindle abnormalities and chromosome misalignment, alteration of maternal mRNAs and delay in pronucleus formation, 2) decreased mitochondrial membrane potential (MMP) and lower adenosine triphosphate (ATP) levels, increased ROS production and DNA damage, G1/S and S/G2 phase transition delay, and delayed first cleavage, and 3) increased early apoptosis and lower levels of cleavage and blastocyst formation. Our results further revealed that such negative impacts of oocyte cryopreservation could be alleviated by supplementation of warming, recovery, PA and IVC media with 10- 9 mol/L MT before the embryos moved into the 2-cell stage of development. CONCLUSIONS: MT might promote cell cycle progression via regulation of MMP, ATP, ROS and maternal mRNA levels, potentially increasing the first cleavage of parthenogenetic zygotes developed from vitrified-warmed mouse oocytes and their subsequent development.

Effect of early pregnancy on the expression of progesterone receptor and progesterone-induced blocking factor in ovine lymph node.

Lymph nodes are the sites where the immune reaction or suppression takes place. Progesterone (P4) exerts an essential effect of the immunomodulation on the maternal uterus during early pregnancy in ruminants. At present study, the inguinal lymph nodes were obtained at day 16 of non-pregnancy, days 13, 16 and 25 of pregnancy (n = 3 for each group) in ewes, and RT-PCR assay, western blot and immunohistochemistry analysis were used to analyze to the effect of early pregnancy on the expression of P4 receptor (PGR) and progesterone-induced blocking factor (PIBF) in the lymph nodes. Our results showed that the PGR and PIBF mRNA were up-regulated in the lymph nodes in pregnant ewes, and the PGR isoform (60 kDa) and the PIBF variant (75 kDa) were expressed constantly in the lymph nodes. However, there was no expression of the PGR isoform (40 kDa) and the PIBF variant (48 kDa) at day 16 of the estrous cycle. The immunohistochemistry results confirmed that the PGR and PIBF proteins were limited to the subcapsular sinus and trabeculae in the cortex, medullary sinuses, and were localized in the cytoplasm of the specific cells. This paper reports for the first time that early pregnancy exerts its effect on the specific cells in the lymph nodes through P4, which results in the up-regulated expression of the PGR mRNA and 40 kDa isoform, the PIBF mRNA and 48 kDa variant, and is involved in the immunoregulation of the lymph nodes through a cytosolic pathway in ewes.