Role of miRNAs in assisted reproductive technology.

Cellular proteins and the mRNAs that encode them are key factors in oocyte and sperm development, and the mechanisms that regulate their translation and degradation play an important role during early embryogenesis. There is abundant evidence that expression of microRNAs (miRNAs) is crucial for embryo development and are highly involved in regulating translation during oocyte and early embryo development. MiRNAs are a group of short (18-24 nucleotides) non-coding RNA molecules that regulate post-transcriptional gene silencing. The miRNAs are secreted outside the cell by embryos during preimplantation embryo development. Understanding regulatory mechanisms involving miRNAs during gametogenesis and embryogenesis will provide insights into molecular pathways active during gamete formation and early embryo development. This review summarizes recent findings regarding multiple roles of miRNAs in molecular signaling, plus their transport during gametogenesis and embryo preimplantation.

Beneficial effects of trehalose and gentiobiose on human sperm cryopreservation.

The protection of human sperm during cryopreservation is of great importance to infertility. Recent studies have shown that this area is still a long way from its ultimate aim of maintaining the maximum viability of sperm in cryopreservation. The present study used trehalose and gentiobiose to prepare the human sperm freezing medium during the freezing-thawing. The freezing medium of sperm was prepared with these sugars, and the sperm were then cryopreserved. The viable cells, sperm motility parameters, sperm morphology, membrane integrity, apoptosis, acrosome integrity, DNA fragmentation, mitochondrial membrane potential, reactive oxygen radicals, and malondialdehyde concentration was evaluated using standard protocols. A higher percentage of the total and progressive motility, rate of viable sperm, cell membrane integrity, DNA and acrosome integrity, and mitochondrial membrane potential were observed in the two frozen treatment groups compared to the frozen control. The cells had less abnormal morphology due to treatment with the new freezing medium than the frozen control. The higher malondialdehyde and DNA fragmentation were significantly observed in the two frozen treatment groups than in the frozen control. According to the results of this study, the use of trehalose and gentiobiose in the sperm freezing medium is a suitable strategy for sperm freezing to improve its motion and cellular parameters.

Determining the Optimal Dosage of Lecithin Nanoliposome in Rooster Semen Freezing Medium and Fertility Potential.

Introduction: Lecithin nanoliposome (nano-LPO), with its cryoprotective properties, is considered to enhance the performance of a traditional semen cryoprotectant. Objective: To determine the optimal dose of lecithin nano-LPO added to the rooster semen extender. Materials and Methods: Semen samples collected weekly from eight broiler breeder roosters were mixed and aliquoted into five equal subsamples, during the five successive weeks. The subsamples were then diluted with a semen extender containing 0%, 0.5%, 1%, 1.5%, or 2% of lecithin nano-LPO. Post-thawed semen quality attributes, including sperm motility and velocity parameters, plasma membrane functionality, mitochondrial membrane potential (MMP), apoptosis-like changes, and fertility potential, were evaluated. Results: Total motility and velocity parameters, including curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity µm/s (VAP), straightness (STR), linearity (LIN), lateral head displacement (ALH), and wobble (WOB) were quadratically (p < 0.01) influenced by graded levels of lecithin nano-LPO, such that the highest values were obtained when 1% of lecithin nano-LPO was used. Treatments had no significant effect on plasma membrane functionality; however, MMP (p < 0.08) and percentages of live and dead spermatozoa (p < 0.05) quadratically responded to increasing levels of lecithin nano-LPO, where the best outcome was found when about 1% of lecithin nano-LPO was used in the semen extender. The percentage of apoptotic spermatozoa cubically responded to increasing levels of lecithin nano-LPO (p ≤ 0.07). No significant trend of fertility rate was found in response to addition of lecithin nano-LPO levels. Conclusions: Supplementing an extender with 1.10% of lecithin nano-LPO is shown to be the optimal dose associated with the most improvement in post-thawed rooster sperm velocity measurements.

Supplementation of freezing medium with encapsulated or free glutathione during cryopreservation of bull sperm.

The objective was to compare effects of encapsulated or free glutathione (GSH) on the quality of frozen-thawed bull sperm. Ejaculates were collected via artificial vagina from six mature Holstein bulls once weekly for 6 weeks. All ejaculates had motility ≥70%, sperm concentration ≥1.0 × 109 /ml and ≤15% morphologically abnormal sperm. Each week, semen was pooled and diluted with lecithin-based extenders containing various concentrations of encapsulated (E0, E1, E2.5 and E5 mM) or free (F0, F1, F2.5 and F5 mM) GSH, with total glutathione content determined before and after cryopreservation. Total GSH in fresh semen was (mean+SEM) 4.8 ± 0.2 nmol/108 sperm, whereas in frozen-thawed semen of group F0 (control), it decreased to 1.4 ± 0.2 nmol/108 sperm, a 70.8% reduction (p < .05). In addition, total GSH in frozen-thawed semen from groups E2.5, E5 and F5 were 2.4 ± 0.2, 2.8 ± 0.2 and 1.8 ± 0.2 nmol/108 sperm, respectively (E5 versus. F0, p < .05). Compared to group F0, frozen-thawed sperm from group E2.5 had greater (p < .05) percentages of sperm that were viable (Annexin-V) (61.1 ± 1.8 versus. 71.1 ± 1.8) and that had cell membrane integrity (eosin-nigrosin) (64.5 ± 3.1 versus. 80.0 ± 3.1). Furthermore, frozen-thawed sperm from group E2.5 had the numerically highest total and progressive motility (CASA) and cell membrane functionality (HOS) and the lowest percentage of early apoptotic sperm (Annexin-V). However, acrosome membrane integrity (PSA) of E5 had the lowest mean (p < .05), whereas E2.5 caused a small nonsignificant decrease (69.1 ± 1.4%) compared to E0 and F0. In conclusion, 2.5 mM encapsulated GSH in semen extender significantly improved the quality of frozen-thawed bull sperm.

Supplementation of the BIOXcell extender with the antioxidants crocin, curcumin and GSH for freezing bull semen.

Semen cryopreservation is routine in cattle, but the results of artificial insemination need improvement. A strategy to these aims is the supplementation of the freezing extender with novel antioxidants. This study aimed at testing the natural antioxidants curcumin and crocin as supplements to the commercial extender BIOXcell for freezing semen from 8 Holstein bulls. We tested curcumin at 0.05 and 0.1 mM (CU0.05, CU0.1) and crocin at 0.5 and 1.5 mM (CR0.5, CR1.5), with 0.5 mM reduced glutathione (GSH0.5) as reference, and a control (CTL, without supplementation). The samples were evaluated post-thawing and after 5 h at 38 °C by CASA for motility and flow cytometry for viability, apoptotic, capacitation, acrosomal status, cytoplasmic and mitochondrial reactive oxygen species (ROS) production, and chromatin status (SCSA). Control and GSH0.5 showed similar results, possibly because of the good protection from BIOXcell. CU0.05 and CU0.1 showed little effects but increased cytoplasmic ROS production and motility ALH. CR0.5 and CR1.5 decreased viability and increased apoptotic features significantly post-thawing and after the incubation, resulting in lower motility (significant after the incubation) but decreasing SCSA %HDS (loose chromatin). Whereas crocin at these concentrations seems incompatible with BIOXcell, maybe because of a prooxidant activity, curcumin use merits further research, considering the elevation of ROS with no significant negative effects.