Extracellular vesicles (EVs) are implicated in various functions within the complex mechanisms of intercellular communication. There are several subpopulations of EVs, including apoptotic bodies, microvesicles and exosomes. These nanovesicles are capable of transferring functional proteins and genetic information to alter the phenotype and function of recipient cells. In animal reproduction, numerous studies have demonstrated that EVs are actively involved in the regulation of different physiological events, modulating a variety of processes such as follicular development, spermatogenesis, oocyte maturation, fertilization and embryo development, with results indicating improved gamete quality, embryo development and cryotolerance. Additionally, EVs show therapeutic potential in restoring reproductive function and supporting maternal-embryonic communication in both domestic and wild species. Therefore, the present review aimed to describe the main studies conducted using EVs in the field of animal reproduction, highlighting their biological relevance, experimental applications and future prospects for clinical implementation in assisted reproductive technologies.

Centromeres are chromosomal loci essential for the correct segregation of genetic material during cell division. Defects in centromere function can lead to aneuploidy and cancer. During early embryonic development in mammals, prior to the first cell division, male and female genomes are separated in pronuclei located at the centre of the zygote. Parental chromatin clusters at the interface between the two pronuclei and this clustering step is critical to avoid aneuploidy in human and bovine zygotes. Yet, despite their essential function in chromosome segregation, the position and spatial organization of centromeres during the first cell cycle in mammals is mostly unknown. Previous studies conducted in bovine embryos derived from in vitro fertilization (IVF) showed that cell cycle progression impacts on the success rate of blastocyst formation. Specifically, embryos that entered earliest into S-phase or the earliest cleaving embryos were more likely to develop into blastocysts. To determine the precise timing of these events we performed a detailed characterization of key phases of the first cell cycle in bovine zygotes derived from IVF. In parallel we examined the spatial positioning of centromeres. We identify 20 h post insemination (hpi) as the timepoint when male and female pronuclei are juxtaposed and are completing S-phase. At this timepoint, we show that centromeres are positioned distal to the pronuclear interface and use super resolution microscopy to demonstrate extensive centromere clustering into chromocentres. Our results identify distinct nuclear features observed at 20 hpi, which may serve as cell cycle markers in determining successful bovine IVF.

Kisspeptin, encoded by Kiss1 gene, regulates reproduction via the hypothalamic-pituitary-gonadal axis. While kisspeptin treatment promotes follicular development in Tan sheep, its direct action on ovarian granulosa cells remains unclear. For this, we first detected the expression of Kiss1 and its receptor Kiss1r in primary ovarian granulosa cells of Tan sheep. Second, the effect of kisspeptin on steroid hormone secretion, proliferation and apoptosis of ovarian granulosa cells was investigated. Third, the signaling pathway of kisspeptin regulating steroid hormone secretion was revealed by western bolting in ovarian granulosa cells. The results showed that the Kiss1 and Kiss1r genes were present in ovarian granulosa cells of Tan-sheep, and 500 nM dose of kisspeptin significantly stimulated steroids hormone secretion (P < 0.05), and up-regulated StAR, HSD17B2, CPY19A1, FSHR, LHR, ERβ, PGR and p-ERK1/2 proteins expression (P < 0.05). Moreover, this treatment significantly promoted cell proliferation and increased the proportion of cells in S phase (P < 0.05), and significantly suppressed granulosa cell apoptosis (P < 0.05). Additionally, the stimulatory effects of kisspeptin on estradiol and progesterone secretion were blocked by inhibitors of the Mitogen-activated Protein Kinase (MAPK) signaling pathway (including PKA inhibitor, PLC inhibitor, PKC inhibitor, and Ca2+inhibitor). Western blot analysis confirmed that kisspeptin regulates steroid hormone secretion primarily through the MAPK-ERK signaling pathway. Our results demonstrate that kisspeptin can directly act on ovarian granulosa cells to promote steroidogenesis, proliferation, and inhibit apoptosis, providing a foundational basis for developing novel kisspeptin-mediated techniques to regulate reproduction in Tan sheep.

Intracytoplasmic sperm injection (ICSI) is a widely used assisted reproduction technique, but in cattle it faces major challenges due to inefficient oocyte activation after sperm microinjection. This study investigated different oocyte activation strategies and assessed the potential role of reducing agents glutathione (GSH), cysteamine (Cys) and dithiobutylamine (DTBA) to improve sperm head decondensation and embryo development following Piezo-ICSI. Haploid parthenogenetic activation using different ethanol concentrations (1%, 3%, 7% and 10%) failed to yield blastocysts, while diploid activation with ethanol or ionomycin combined with inhibitors significantly improved cleavage (43–55%) and blastocyst rates (14–27%), respectively. However, applying two ethanol pulses was detrimental, reducing both cleavage and blastocysts likely due to toxic overexposure. Sperm head decondensation compounds in Piezo-ICSI showed a high percentage of inactivated oocytes (75% GSH, 55% Cys and 40% DTBA). The highest male pronuclear formation rates were observed in the control without sperm head decondensation (21%) and with DTBA treatment (10%). Despite this, the treatment with Cys resulted in higher developmental potential to the blastocyst stage (22%) comparable to the control (24%). These data suggest that the inclusion of sperm head decondensing agents could represent a promising new strategy for enhancing the early in vitro development of ICSI-generated embryos. However, for this purpose, careful optimization of the concentration and incubation time of these decondensing compounds is essential.