Università degli Studi di Milano, Dipartimento di Medicina Veterinaria e Scienze Animali (DIVAS), Lodi, Italy.
Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche IBBA CNR, Lodi, Italy.
Theriogenology. 2022 Dec;194:35-45. doi: 10.1016/j.theriogenology.2022.09.023. Epub 2022 Sep 28.
Unlike humans and many other mammalian species, conventional in vitro fertilization (IVF) in equine species is not successful. To mimic in vitro equine spermatozoon-oviduct interaction as close as possible to that which occurs in vivo, extracellular vesicles (EVs) secreted by the female genital tract were used. Three female genital tracts were collected at slaughterhouse from mares in late estrus. Ipsilateral proximal and apical horn endometrial explants were digested with collagenase and trypsin and cells obtained were cultured on insert system to allow their polarization. Ipsilateral oviducts were squeezed out to obtain spheroids. To produce EVs, proximal and apical horn endometrial cells and oviductal spheroids were cultured for three days in serum free medium. To trace interaction between spermatozoa and EVs by fluorescence microscopy, EVs were differently labeled. Pooled samples of ejaculated spermatozoa from three stallions were incubated in capacitating medium (CM) for 6 h and to induce hyperactivation for other 6 h in CM supplemented with different kind of EVs alone or in combination. A control was performed in absence of EVs. Sperm were assessed for motility by CASA system, EV incorporation by confocal microscopy and acrosomal reaction (AR) by staining with FITC-PNA/PI. In vitro fertilization was performed, and presumed zygotes were subjected to chromatin configuration. The results show that incorporation of EVs of the proximal horn does not take place, while apical horn EVs are incorporated in the head of the spermatozoon in 4 h. The EVs of oviductal spheroids are incorporated in the middle tract in 1 h. The rate of AR with EVs of the apical horn and oviductal spheroids were respectively 50.25% and 57.14%. When these EVs were added in combination, the rate of AR was 71.42%. In the control, the rate of AR was of 15%. After in vitro fertilization, 44% of oocytes showed male and female pronuclei, whereas no fertilization is obtained in the control. In conclusion, EVs from apical horn and oviduct could be involved in cell trafficking during equine semen hyperactivation, and their possible use in vitro could facilitate the development of equine reproductive biotechnologies.
与人类和许多其他哺乳动物物种不同，马属动物的常规体外受精（IVF）并不成功。为了尽可能模拟体内发生的体外马精子-输卵管相互作用，使用了雌性生殖道分泌的细胞外囊泡（EVs）。在屠宰场从发情后期的母马中收集了三个雌性生殖道。用胶原酶和胰蛋白酶消化同侧近端和顶端角子宫内膜外植体，并在插入系统上培养细胞以允许其极化。挤出同侧输卵管以获得球体。为了产生 EVs，将近端和顶端角子宫内膜细胞和输卵管球体在无血清培养基中培养三天。为了通过荧光显微镜追踪精子和 EVs 之间的相互作用，EVs 被不同地标记。从三头种马中收集的精子混合样本在获能培养基（CM）中孵育 6 小时，并在补充有不同类型 EVs 的 CM 中进一步诱导超激活 6 小时，单独或组合使用。在没有 EVs 的情况下进行对照。通过 CASA 系统评估精子的运动能力，通过共聚焦显微镜评估 EV 的掺入情况，并通过 FITC-PNA/PI 染色评估顶体反应（AR）。进行体外受精，并对假定的受精卵进行染色质构型分析。结果表明，近端 horn 的 EVs 不被掺入，而顶端 horn 的 EVs 在 4 小时内被掺入精子头部。输卵管球体的 EVs 在 1 小时内被掺入中段。用顶端 horn 和输卵管球体的 EVs 诱导的 AR 率分别为 50.25%和 57.14%。当这些 EVs 联合使用时，AR 率为 71.42%。在对照组中，AR 率为 15%。体外受精后，44%的卵母细胞显示出雌雄原核，而在对照组中未获得受精。总之，来自顶端 horn 和输卵管的 EVs 可能参与马精液超激活过程中的细胞运输，并且它们在体外的可能应用可能有助于马生殖生物技术的发展。
