Search Results (16)

Fertilisation failure following intracytoplasmic sperm injection (ICSI) is a significant challenge in assisted reproductive technology (ART), particularly in the absence of an identifiable cause. Artificial oocyte activation (AOA), typically with calcium ionophores, has emerged as a potential solution in scenarios characterised by a deficiency of phospholipase C zeta (PLCζ). This narrative review consolidates the latest clinical and experimental data regarding the application of calcium ionophores for oocyte activation, the significance of PLCζ testing in instances of unexplained fertilisation failure, and the impact of AOA on the morphokinetics and developmental potential of embryos. AOA has demonstrated an enhancement in fertilisation, cleavage, and pregnancy outcomes in specific patient populations, including individuals with diminished ovarian reserve or those who have previously attempted conception unsuccessfully. Although AOA appears to have no impact on embryo morphokinetics, certain studies indicate slight alterations in early cleavage features. The available statistics indicate that there are no significant safety concerns about outcomes for babies. This finding underscores the significance of tailored ART methodologies that incorporate molecular diagnostics and targeted AOA therapies. It emphasises the necessity for additional prospective trials to enhance patient selection and long-term safety surveillance. Full article

The successful application of assisted reproductive technologies (ARTs), such as in vitro maturation (IVM) and artificial oocyte activation, requires species-specific adaptations. Although these methods are routinely used in laboratory rodents, their use in wild or non-model species remains limited, such as the Spix’s yellow-toothed cavy, a Neotropical species of ecological and reproductive interest. This study evaluated the effects of different concentrations of epidermal growth factor (EGF; 10 or 50 ng/mL) on IVM (Experiment 1) and of 6-dimethylaminopurine (6-DMAP) on artificial oocyte activation (Experiment 2). EGF at 10 ng/mL (93.8% ± 1.6; 84.9% ± 0.7) promoted greater viability and less apoptosis in cumulus cells, compared to 50 ng/mL (83.0% ± 1.6; 78.9% ± 2.7), maintaining cumulus expansion, ultrastructural integrity, and better morphometric quality of oocytes. Thus, this concentration was used in the next step, where oocytes were activated with or without 6-DMAP. After five days, the presence of 6-DMAP increased cleavage rates (69.3% ± 5.0) compared to activation without the compound (53.5% ± 3.5), without significantly affecting morula formation (13.2% ± 3.1 to 17.3% ± 2.9). It is concluded that EGF improves the oocyte microenvironment, while 6-DMAP enhances cleavage, with these being the initial steps in the development of ARTs for Spix’s yellow-toothed cavy. Full article

Advancements in assisted reproductive technologies (ARTs) have led to the development of various add-on techniques aimed at improving oocyte quality and enhancing embryo implantation potential. These techniques target critical stages of both oocyte and embryo physiology, including oocyte growth and maturation, fertilization, chromosomal status, and embryo development. Key approaches involve the optimization of in vitro fertilization (IVF) protocols, recruiting capable follicles giving rise to dynamic oocytes to evolve, culture media supplementation, preimplantation genetic testing (PGT), and mitochondrial replacement therapy (MRT), all of which are designed to enhance oocyte competence through its function and metabolism. The use of PGT has been promising in selecting embryos suitable for transfer, thus optimizing implantation success. Emerging technologies, such as platelet-rich plasma treatment (PRP), time-lapse imaging (TLI), and hyaluronan-rich (HA) culture media, claim to improve ovarian rejuvenation and uterine receptivity, embryo selection, as well as embryo implantation potential, respectively. Evidence for certain add-on approaches remains limited, but ongoing research suggests that the use of such treatments may lead to increased clinical pregnancies and live birth rates, especially in poor-prognosis patients. The present review describes the current state of the add-on innovations, their mechanisms of action, as well as their possibilities to increase ART success rates. Full article

Antioxidants protect cellular function and structure by neutralizing the oxidative stress caused by increased reactive oxygen species (ROS) during sperm freezing. Studies on cryopreservation using various antioxidants have demonstrated encouraging results. Many studies have used antioxidants to increase the efficiency of sperm freezing and to improve the success rate of artificial insemination and pregnancy. Manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) is a newly synthesized antioxidant with positive effects on sperm morphology and capacitation in humans, rams, and stallions. In this study, porcine semen was treated with 0, 50, 100, and 150 μM of MnTBAP based on a Tris–egg-yolk extender and frozen to determine whether MnTBAP can assist the status of sperm during cryopreservation. First, motility was assessed using the computer-assisted sperm analysis (CASA) system, with the 100 μM treatment group showing the highest motile rate (66.8%) compared with that of the other groups (control, 51.1%; 50 μM and 150 μM, 59.6%); therefore, the remaining analyses were conducted comparing the two groups (control vs. 100 μM group; p < 0.01). Second, fluorescence staining was applied to examine the control and 100 μM groups using fluorescence microscopy. The viability (41.7% vs. 62.4%) and the acrosome integrity (77.9% vs. 86.4%) differed significantly (p < 0.05). In addition, the mitochondrial membrane potential (MMP) was 46.5% vs. 51.9%; the fragmentation rate, estimated using the Sperm-sus-Halomax kit, was 63.4% vs. 57.4%; and the detected caspase activity was 30.1% vs. 22.9%. These tended to be higher in the treated group but did not differ significantly. Third, measurements using FACSLyric revealed that the 100 μM treatment group exhibited a state of elevated normal lipid arrangement within the plasma membrane and diminished levels of apoptosis and ROS (p < 0.01). We assessed the expression of genes relevant to antioxidant effectiveness using real-time RT-qPCR. Our findings indicated significant alterations in the expression levels of various mRNA species, with the exception of NOX5 (p < 0.05). Finally, the straws were dissolved and used to treat matured denuded oocytes to investigate the effect on fertilization and embryo development in vitro. The cleavage rate was (77.6% vs. 84.1%), and the blastocyst rate was 9.7% vs. 11.4% (p < 0.05). In conclusion, these results suggest that MnTBAP positively affected sperm freeze–thawing, improving the fertilization capacity, and leading to increased embryo development. Full article

Postovulatory aging is known to impair the oocyte quality and embryo development due to oxidative stress in many different animal models, which reduces the success rate or pregnancy rate in human assisted reproductive technology (ART) and livestock timed artificial insemination (TAI), respectively. Salidroside (SAL), a phenylpropanoid glycoside, has been shown to exert antioxidant and antitumor effects. This study aimed to investigate whether SAL supplementation could delay the postovulatory oocyte aging process by alleviating oxidative stress. Here, we show that SAL supplementation decreases the malformation rate and recovers mitochondrial dysfunction including mitochondrial distribution, mitochondrial membrane potential (ΔΨ) and ATP content in aged oocytes. In addition, SAL treatment alleviates postovulatory aging-caused oxidative stress such as higher reactive oxygen species (ROS) level, lower glutathione (GSH) content and a reduced expression of antioxidant-related genes. Moreover, the cytoplasmic calcium ([Ca²⁺]c) and mitochondrial calcium ([Ca²⁺]mt) of SAL-treated oocytes return to normal levels. Notably, SAL suppresses the aging-induced DNA damage, early apoptosis and improves spindle assembly in aged oocytes, ultimately elevating the embryo developmental rates and embryo quality. Finally, the RNA-seq and confirmatory experience showed that SAL promotes protective autophagy in aged oocytes by activating the MAPK pathway. Taken together, our research suggests that supplementing SAL is an effective and feasible method for preventing postovulatory aging and preserving the oocyte quality, which potentially contributes to improving the successful rate of ART or TAI. Full article

Egg activation is a cellular transition of an arrested mature oocyte into a developing embryo through a coordinated series of events. Previous studies in Hymenoptera have indicated that mechanical pressure can induce egg activation. In this study, we developed the first egg activation protocol for the haplodiploid insect pest, Sirex noctilio (Hymenoptera: Siricidae), from two climatically different regions in South Africa to demonstrate the broad applicability of the method. In addition, activated eggs were exposed to three treatments involving water, pine sawdust, and the fungal symbiont of S. noctilio, Amylostereum areolatum (Russulales: Amylostereaceae), to determine if the symbiotic fungus is a requirement for egg development in an artificial laboratory environment, as the symbiotic fungus has been hypothesised to be necessary for egg and early larval development in a natural environment. A rearing protocol was developed for the first instar larvae using a modified Anoplophora glabripennis (Coleoptera: Cerambycidae) artificial diet. A significant difference between the mean survival rates of activated eggs from the two different regions was observed. Amylostereum areolatum was shown to be unnecessary for egg survival and adversely affected egg eclosion in an artificial laboratory environment. The maximum larval survival duration on the artificial diet was 92 days. The egg activation and rearing protocol developed in this study enables opportunities for research on the physiology, ecology, symbioses, and genetics of S. noctilio, which can be exploited for new genetic pest management strategies. Full article

During mammalian fertilization, repetitive rises of intracellular calcium called calcium oscillations are required for full activation of oocytes. Therefore, oocytes such as round spermatid injected or somatic cell nuclear transferred require additional artificial activation which mimics the calcium oscillations. It is well recognized that sperm specific phospholipase C (PLCζ) is a strong candidate as the sperm factor which can induce calcium oscillations and, at least in mammals, the genetic mutation of PLCζ in human causes male infertility due to the lack of calcium oscillations in the oocytes. Recent studies showed that the sperm lacking PLCζ (Plcz1^−/−) still could induce rise(s) of intracellular calcium in the oocytes after IVF but not intracytoplasmic sperm injection (ICSI). In the ICSI oocytes, no pronuclear formation or development to the two-cell stage was observed. However, it is still unclear whether additional activation treatment can rescue the low developmental ability of Plcz1^−/−-sperm-derived oocytes after ICSI. In this study, we examined whether oocytes injected with a Plcz1^−/− sperm can develop to term by additional artificial activation. In oocytes injected a Plcz1^−/− sperm and Plcz1^−/− and eCS (another candidate of the sperm factor) double knockout sperm (Plcz1^−/−eCS^−/−), the rates of pronuclear formation were very low (2.0 ± 2.3% and 6.1 ± 3.7%, respectively) compared to control (92.1 ± 2.6%). However, these rates were dramatically improved by additional procedures of PLCζ-mRNA injection or SrCl₂ treatment (Plcz1^−/− sperm + PLCζ mRNA, Plcz1^−/− sperm + SrCl₂ and Plcz1^−/−eCS^−/− sperm + PLCζ mRNA; 64.2 ± 10.8%, 89.2 ± 2.4% and 72.6 ± 5.4%, respectively). Most of the oocytes were developed to the two-cell stage. After embryo transfer, healthy pups were obtained in all these groups (Plcz1^−/− sperm + PLCζ mRNA:10.0 ± 2.8%, Plcz1^−/− sperm + SrCl₂:4.0 ± 4.3% and Plcz1^−/−eCS^−/− sperm + PLCζ mRNA: 10.0 ± 5.7%). The rate in Plcz1^−/− sperm + SrCl₂ group was significantly lower than that in control (26.0 ± 2.4%). Taken together, our present results show that additional activation treatment such as SrCl₂ and PLCζ mRNA can fully support to develop to term even in oocyte injected Plcz1^−/− sperm. In addition, PLCζ-induced oocyte activation is more suitable for successful development to term compared to that such as phenomenon induced by SrCl₂. These findings will contribute to improvement for male-dependent human infertility and reproductive technologies in other mammalian species. Full article

The glucosinolate transporters 1/2/3 (GTR1/2/3) from the Nitrate and Peptide transporter Family (NPF) play an essential role in the transport, accumulation, and distribution of the specialized plant metabolite glucosinolates. Due to representing both antinutritional and health-promoting compounds, there is increasing interest in characterizing GTRs from various plant species. We generated seven artificial glucosinolates (either aliphatic or benzenic) bearing different fluorophores (Fluorescein, BODIPY, Rhodamine, Dansylamide, and NBD) and investigated the ability of GTR1/2/3 from Arabidopsis thaliana to import the fluorescent glucosinolates (F-GSLs) into oocytes from Xenopus laevis. Five out of the seven F-GSLs synthesized were imported by at least one of the GTRs. GTR1 and GTR2 were able to import three F-GSLs actively above external concentration, while GTR3 imported only one actively. Competition assays indicate that the F-GSLs are transported by the same mechanism as non-tagged natural glucosinolates. The GTR-mediated F-GSL uptake is detected via a rapid and sensitive assay only requiring simple fluorescence measurements on a standard plate reader. This is highly useful in investigations of glucosinolate transport function and provides a critical prerequisite for elucidating the relationship between structure and function through high-throughput screening of GTR mutant libraries. The F-GSL themselves may also be suitable for future studies on glucosinolate transport in vivo. Full article

After sperm-oocyte fusion, intracytoplasmic rises of calcium (Ca) induce the release of zinc (Zn) out of the oocyte (Zn sparks). Both phenomena are known to play an essential role in the oocyte activation process. Our work aimed to explore different protocols for activating bovine and porcine oocytes using the novel zinc chelator 1,10-phenanthroline (PHEN) and to compare developmental rates and quality to bovine IVF and parthenogenetic ionomycin-induced embryos in both species. Different incubation conditions for the zinc chelator were tested, including its combination with ionomycin. Embryo quality was assessed by immunofluorescence of SOX2, SOX17, OCT4, and CDX2 and total cell number at the blastocyst stage. Even though blastocyst development was achieved using a zinc chelator in bovine, bypassing calcium oscillations, developmental rates, and blastocyst quality were compromised compared to embryos generated with sperm-induced or ionomycin calcium rise. On the contrary, zinc chelation is sufficient to trigger oocyte activation in porcine. Additionally, we determined the optimal exposure to PHEN for this species. Zinc chelation and artificial induction of calcium rise combined did not improve developmental competence. Our results contribute to understanding the role of zinc during oocyte activation and preimplantation embryo development across different mammalian species. Full article

The greater amberjack (Seriola dumerili), a pelagic marine species with a global distribution, has considerable worldwide potential as an aquaculture species. However, difficulties have been encountered in inducing spontaneous spawning in cultured fish stocks. In this study, we analysed the key regulatory factors, secretoneurin (SN) and gonadotropin-releasing hormone (GnRH), in greater amberjack. Active peptides of SN and GnRH, SdSNa, and SdGnRH, respectively, were obtained by comparative analysis of homologous proteins from different species. Amino acid substitutions of the SdGnRH decapeptide at position 6 with a dextrorotatory (D) amino acid and at position 10 with an ethylamide group yielded a super-active agonist (SdGnRHa). The injection of SdSNa and SdGnRHa elevated luteinizing hormone, thyroid-stimulating hormone, and oxytocin levels in the sera of sexually mature fish, whereas it reduced the level of follicle-stimulating hormone. Furthermore, in response to the SdSNa and SdGnRHa injections, we detected an increase in the expression of genes associated with oocyte development and spermatogenesis. We established that the greater amberjack cultured along the southern coast of China reached sexual maturity at three years of age, and its reproductive season extended from February to April. Spawning of the cultured greater amberjack was successfully induced with a single injection of SdGnRHa/SdSN/DOM/HCG. Our findings indicate that similar to GnRHa, SNa is a potential stimulator of reproduction that can be used to artificially induce spawning in marine fish. Full article

Artificial directional selection has replaced natural selection and resulted in trait differences across breeds in domestic animal breeding. However, the molecular mechanism by which the oviduct regulates litter size remains largely elusive in goats during the follicular phase. Accumulating data have linked lncRNAs to reproductive activities; however, little is known about the modulation mechanism in the oviduct. Herein, RNA-seq was used to measure mRNA and lncRNA expression levels in low- and high-fecundity goats. We observed distinctive differences in mRNA and lncRNA in terms of different kidding numbers and detected the differential expression of 1640 mRNA transcripts and 271 lncRNA transcripts. Enrichment analysis of differentially expressed mRNAs (DEGs) suggested that multiple pathways, such as the AMPK, PI3K–Akt, calcium signaling pathway, oocyte meiosis, ABC transporter, and ECM–receptor interaction pathways, directly or indirectly affected goat reproduction. Additionally, coexpression of differentially expressed lncRNAs (DEL)-genes analysis showed that XLOC_021615, XLOC_119780, and XLOC_076450 were trans-acting as the DEGs ATAD2, DEPDC5, and TRPM6, respectively, and could regulate embryo development. Moreover, XLOC_020079, XLOC_107361, XLOC_169844, XLOC_252348 were the trans-regulated elements of the DEGs ARHGEF2 and RAPGEF6, and the target DEGs CPEB3 of XLOC_089239, XLOC_090063, XLOC_107409, XLOC_153574, XLOC_211271, XLOC_251687 were associated with prolificacy. Collectively, our study has offered a thorough dissection of the oviduct lncRNA and mRNA landscapes in goats. These results could serve as potential targets of the oviduct affecting fertility in goats. Full article

The purpose of this study was to investigate the periodic seasonal changes in endocrine activity and gonadal development of female rainbow trout (Oncorhynchus mykiss) in a high-altitude cold-water environment. The fish were sampled monthly from January to November and the levels of plasma hormones (estradiol (E₂), cortisol and thyroid hormones (TH_S)) and vitellogenin (VTG) were measured by ELISA. Moreover, the transcriptions of sex-related genes in the ovary, brain, and liver were detected by qRT-PCR. The results showed a seasonal fluctuation of plasma hormones and VTG together with the development of the ovary, which reached a peak from August to October. Similarly, the transcription of hypothalamic gonadotropin-releasing hormone-2 (cgnrh-2), hypothalamic gonadotropin-releasing hormone receptors (gnrhr) and follicle-stimulating hormone (fsh) in the brain varied from January to September, but the highest level was detected in September to November. In addition, the transcription of sex-related genes located in the ovary and liver increased significantly during August to October, accompanied by a continuous increase in the gonadosomatic index (GSI) and a decrease in the hepatosomatic index (HSI). Therefore, plasma hormones and sex-related genes regulate the development and maturation of O. mykiss oocytes with the change in seasons and peaked in November. The results of this study provide a reference for improving the efficiency of the artificial reproduction of O. mykiss. Full article

Parthenogenesis activation (PA), as an important artificial breeding method, can stably preserve the dominant genotype of a species. However, the delayed development of PA embryos is still overly severe and largely leads to pre-implantation failure in pigs. The mechanisms underlying the deficiencies of PA embryos have not been completely understood. For further understanding of the molecular mechanism behind PA embryo failure, we performed transcriptome analysis among pig oocytes (meiosis II, MII) and early embryos at three developmental stages (zygote, morula, and blastocyst) in vitro fertilization (IVF) and PA group. Totally, 11,110 differentially expressed genes (DEGs), 4694 differentially expressed lincRNAs (DELs) were identified, and most DEGs enriched the regulation of apoptotic processes. Through cis- and trans-manner functional prediction, we found that hub lincRNAs were mostly involved in abnormal parthenogenesis embryonic development. In addition, twenty DE imprinted genes showed that some paternally imprinted genes in IVF displayed higher expression than that in PA. Notably, we identified that three DELs of imprinted genes (MEST, PLAGL1, and DIRAS3) were up regulated in IVF, and there was no significant change in PA group. Disordered expression of key genes for embryonic development might play key roles in abnormal parthenogenesis embryonic development. Our study indicates that embryos derived from different production techniques have varied in vitro development to the blastocyst stage, and they also affect the transcription level of corresponding genes, such as imprinted genes. This work will help future research on these genes and molecular-assisted breeding for pig parthenotes. Full article

Artificial activation of oocytes is an important step for successful parthenogenesis and somatic cell nuclear transfer (SCNT). Here, we investigated the initiation of DNA synthesis and in vivo development of canine PA embryos and cloned embryos produced by treatment with 1.9 mM 6-dimethylaminopurine (6-DMAP) for different lengths of time. For experiments, oocytes for parthenogenesis and SCNT oocytes were cultured for 4 min in 10 μM calcium ionophore, and then divided into 2 groups: (1) culture for 2 h in 6-DMAP (DMAP-2h group); (2) culture for 4 h in DMAP (DMAP-4h group). DNA synthesis was clearly detected in all parthenogenetic (PA) embryos and cloned embryos incorporated BrdU 4 h after activation in DMAP-2h and DMAP-4h groups. In vivo development of canine parthenogenetic fetuses was observed after embryo transfer and the implantation rates of PA embryos in DMAP-2h were 34%, which was significantly higher than those in DMAP-4h (6.5%, p < 0.05). However, in SCNT, there was no significant difference in pregnancy rate (DMAP-2h: 41.6% vs. DMAP-4h: 33.3%) and implantation rates (DMAP-2h: 4.94% vs. DMAP-4h: 3.19%) between DMAP-2h and DMAP-4h. In conclusion, the use of DMAP-2h for canine oocyte activation may be ideal for the in vivo development of PA zygotes, but it was not more effective in in vivo development of canine reconstructed SCNT oocytes. The present study demonstrated that DMAP-2h treatment on activation of canine parthenogenesis and SCNT could effectively induce the onset of DNA synthesis during the first cell cycle. Full article

Building on our recent discovery of the zinc signature phenomenon present in boar, bull, and human spermatozoa, we have further characterized the role of zinc ions in the spermatozoa’s pathway to fertilization. In boar, the zinc signature differed between the three major boar ejaculate fractions, the initial pre-rich, the sperm-rich, and the post-sperm-rich fraction. These differences set in the sperm ejaculatory sequence establish two major sperm cohorts with marked differences in their sperm capacitation progress. On the subcellular level, we show that the capacitation-induced Zn-ion efflux allows for sperm release from oviductal glycans as analyzed with the oviductal epithelium mimicking glycan binding assay. Sperm zinc efflux also activates zinc-containing enzymes and proteases involved in sperm penetration of the zona pellucida, such as the inner acrosomal membrane matrix metalloproteinase 2 (MMP2). Both MMP2 and the 26S proteasome showed severely reduced activity in the presence of zinc ions, through studies using by gel zymography and the fluorogenic substrates, respectively. In the context of the fertilization-induced oocyte zinc spark and the ensuing oocyte-issued polyspermy-blocking zinc shield, the inhibitory effect of zinc on sperm-borne enzymes may contribute to the fast block of polyspermy. Altogether, our findings establish a new paradigm on the role of zinc ions in sperm function and pave the way for the optimization of animal semen analysis, artificial insemination (AI), and human male-factor infertility diagnostics. Full article