Roles of Key Epigenetic Regulators in the Gene Transcription and Progression of Prostate Cancer

Prostate cancer (PCa) is a top-incidence malignancy, and the second most common cause of death amongst American men and the fifth leading cause of cancer death in men around the world. Androgen receptor (AR), the key transcription factor, is critical for the progression of PCa by regulating a series of target genes by androgen stimulation. A number of co-regulators of AR, including co-activators or co-repressors, have been implicated in AR-mediated gene transcription and PCa progression. Epigenetic regulators, by modifying chromatin integrity and accessibility for transcription regulation without altering DNA sequences, influence the transcriptional activity of AR and further regulate the gene expression of AR target genes in determining cell fate, PCa progression and therapeutic response. In this review, we summarized the structural interaction of AR and epigenetic regulators including histone or DNA methylation, histone acetylation or non-coding RNA, and functional synergy in PCa progression. Importantly, epigenetic regulators have been validated as diagnostic markers and therapeutic targets. A series of epigenetic target drugs have been developed, and have demonstrated the potential to treat PCa alone or in combination with antiandrogens.

Role of Mono- and Disaccharide Combination in Cryoprotective Medium for Rooster Semen to Ensure Cryoresistance of Spermatozoa

The combination of saccharides in the composition of a cryopreservation medium may represent a promising method for the preservation of the reproductive cells of male birds. In the current study, cryoprotective media with a combined composition of mono- and di-saccharides were developed. The degree of penetration of reducing saccharide molecules (maltose—Mal20 medium) and non-reducing disaccharide molecules (trehalose—Treh20 medium) from the cryoprotective medium into the cytosol of rooster spermatozoa was studied. LCM control media without disaccharides were used as the control. The number of maltose molecules penetrating from the outside into the cytosol of the spermatozoon was 1.06 × 104, and the number of trehalose molecules was 3.98 × 104. Using a combination of maltose and fructose, the progressive motility of frozen/thawed semen and the fertility rates of eggs were significantly higher ((p < 0.05) 40.2% and 68.5%, respectively) than when using a combination of trehalose and fructose in a cryoprotective diluent (33.4% and 62.4%, respectively). A higher rate of chromatin integrity at the level of 92.4% was obtained when using Treh20 versus 74.5% Mal20 (p < 0.05). Maltose positively affected the preservation of frozen/thawed sperm in the genital tract of hens. On the seventh day from the last insemination when using Mal20, the fertilization of eggs was 42.6% and only 27.3% when using Treh20. Despite the same molecular weight, maltose and trehalose have different physicochemical and biological properties that determine their function and effectiveness as components of cryoprotective media.

L-Carnitine Improves Cytoprotection during Cryopreservation: A case study on Nili-Ravi Buffalo Sperm

The current study was aimed to evaluate the antioxidative effect of L-Carnitine at post thawing following cryopreservation of Nili-Ravi buffalo sperm. For this purpose, semen from three buffalo bulls were collected for 3 weeks using an artificial vagina (N=18; replicates). The qualified ejaculates were diluted employing tris-citric acid extender i.e., control did not receive any L-Carnitine and experimental groups having 0.5, 1.0, and 1.5 ng/mL of L-carnitine at 37 C with approximately 50 x 106 sperm/mL. The semen was cooled at 4 C and then equilibrated (4 hours), filled in straws (0.5 mL) at4 C, placed on LN2 vapours for 10 min, and kept into an LN2 container. The thawed semen was evaluated for post-thaw quality. The integrity of the sperm plasma membrane and motility (P?0.05) was highest in the extenders having 1.0 ng/mL of L-carnitine as compared to the control(received no L-Carnitine). However, sperm chromatin integrity and viability(live sperm with intact acrosome) remained similar. It was concluded that supplementing 1.0 ng/mL L-Carnitine of extender can improve the post-thaw quality of cryopreserved sperm. Based on the results of the current experiments it is recommended to include L-carnitine extender to improve post-thaw quality of buffalo sperm in terms of its motility and integrity ofits plasma membrane. Keywords: Buffalo, Sperm, Cryopreservation, Extender, L-Carnitine, Artificial insemination.

FACT-mediated maintenance of chromatin integrity during transcription is essential for viability of mammalian stem cells

Replication and transcription machineries access DNA through unwrapping it from histone core. Preservation of nucleosome structure during replication is a focus of intensive research, while maintenance of nucleosomes during transcription is less studied. Histone chaperone FACT is involved in transcription elongation, although whether it opens nucleosomes for polymerase or protects core histones from loss during passage of polymerase is still unclear. We used conditional knockout model of Ssrp1, subunit of FACT complex, to deplete FACT in mice and monitored consequences of FACT loss to establish the functional relevance of FACT in mammals. FACT loss was lethal for mice at all ages due to failure of hematopoietic and intestinal tissues. In these tissues, only the progenitors completely vanished upon FACT loss, while number of some other cell types were changed up and down. Using isolated stem cells of several tissues we showed that FACT loss was toxic only for stem cells, but not for cells which were differentiated in vitro. Chromatin accessibility in stem cells was increased genome wide upon FACT depletion in transcription dependent manner. The most prominent response to the loss of chromatin integrity upon FACT depletion in cells was activation of interferon signaling, followed by accumulation of immune cells in sensitive organs. Thus, in undifferentiated mammalian cells FACT keeps chromatin stable during transcription elongation and this function of FACT is essential for viability of stem cells.

Single-center thorough evaluation and targeted treatment of globozoospermic men

Purpose To characterize, by specific biomarkers and nucleic acid sequencing, the structural and genomic sperm characteristics of partial (PG) and complete globozoospermic (CG) men in order to identify the best reproductive treatment. Methods We assessed spermatozoa from 14 consenting men ultrastructurally, as well as for histone content, sperm chromatin integrity, and sperm aneuploidy. Additional genomic, transcriptomic, and proteomic evaluations were carried out to further characterize the CG cohort. The presence of oocyte-activating sperm cytosolic factor (OASCF) was measured by a phospholipase C zeta (PLCζ) immunofluorescence assay. Couples were treated in subsequent cycles either by conventional ICSI or by ICSI with assisted gamete treatment (AGT) using calcium ionophore (Ionomycin, 19657, Sigma-Aldrich, Saint Louis, MO, USA). Results Ultrastructural assessment confirmed complete acrosome deficiency in all spermatozoa from CG men. Histone content, sperm chromatin integrity, and sperm aneuploidy did not differ significantly between the PG (n = 4) and CG (n = 10) cohorts. PLCζ assessment indicated a positive presence of OASCF in 4 PG couples, who underwent subsequent ICSI cycles that yielded a 36.1% (43/119) fertilization with a 50% (2/4) clinical pregnancy and delivery rate. PLCζ assessment failed to detect OASCF for 8 CG patients who underwent 9 subsequent ICSI cycles with AGT, yielding a remarkable improvement of fertilization (39/97; 40.2%) (P = 0.00001). Embryo implantation (6/21; 28.6%) and clinical pregnancies (5/7; 71.4%) were also enhanced, resulting in 4 deliveries. Gene mutations (DPY19L2, SPATA16, PICK1) were identified in spermatozoa from CG patients. Additionally, CG patients unable to sustain a term pregnancy had gene mutations involved in zygote development (NLRP5) and postnatal development (BSX). CG patients who successfully sustained a pregnancy had a mutation (PIWIL1) related to sperm phenotype. PLCZ1 was both mutated and underexpressed in these CG patients, regardless of reproductive outcome. Conclusions Sperm bioassays and genomic studies can be used to characterize this gamete’s capacity to support embryonic development and to tailor treatments maximizing reproductive outcome.

Effects of Porcine Immature Oocyte Vitrification on Actin Microfilament Distribution and Chromatin Integrity During Early Embryo Development in vitro

Vitrification is mainly used to cryopreserve female gametes. This technique allows maintaining cell viability, functionality, and developmental potential at low temperatures into liquid nitrogen at −196°C. For this, the addition of cryoprotectant agents, which are substances that provide cell protection during cooling and warming, is required. However, they have been reported to be toxic, reducing oocyte viability, maturation, fertilization, and embryo development, possibly by altering cell cytoskeleton structure and chromatin. Previous studies have evaluated the effects of vitrification in the germinal vesicle, metaphase II oocytes, zygotes, and blastocysts, but the knowledge of its impact on their further embryo development is limited. Other studies have evaluated the role of actin microfilaments and chromatin, based on the fertilization and embryo development rates obtained, but not the direct evaluation of these structures in embryos produced from vitrified immature oocytes. Therefore, this study was designed to evaluate how the vitrification of porcine immature oocytes affects early embryo development by the evaluation of actin microfilament distribution and chromatin integrity. Results demonstrate that the damage generated by the vitrification of immature oocytes affects viability, maturation, and the distribution of actin microfilaments and chromatin integrity, observed in early embryos. Therefore, it is suggested that vitrification could affect oocyte repair mechanisms in those structures, being one of the mechanisms that explain the low embryo development rates after vitrification.