Out of millions of ejaculated sperm, a few reach the fertilization site in mammals. Flagellar Ca²⁺signaling nanodomains, organized by multi-subunit CatSper calcium channel complexes, are pivotal for sperm migration in the female tract, implicating CatSper-dependent mechanisms in sperm selection. Here using biochemical and pharmacological studies, we demonstrate that CatSper1 is an O-linked glycosylated protein, undergoing capacitation-induced processing dependent on Ca²⁺and phosphorylation cascades. CatSper1 processing correlates with protein tyrosine phosphorylation (pY) development in sperm cells capacitated in vitro and in vivo. Using 3D in situ molecular imaging and ANN-based automatic detection of sperm distributed along the cleared female tract, we demonstrate that spermatozoa past the utero-tubal junction possess the intact CatSper1 signals. Together, we reveal that fertilizing mouse spermatozoa in situ are characterized by intact CatSper channel, lack of pY, and reacted acrosomes. These findings provide molecular insight into sperm selection for successful fertilization in the female reproductive tract.

Gamete fusion is a critical event of mammalian fertilization. A random one-bead one-compound combinatorial peptide library represented synthetic human egg mimics and identified a previously unidentified ligand as Fc receptor–like 3, named MAIA after the mythological goddess intertwined with JUNO. This immunoglobulin super family receptor was expressed on human oolemma and played a major role during sperm-egg adhesion and fusion. MAIA forms a highly stable interaction with the known IZUMO1/JUNO sperm-egg complex, permitting specific gamete fusion. The complexity of the MAIA isotype may offer a cryptic sexual selection mechanism to avoid genetic incompatibility and achieve favorable fitness outcomes.

Background: Capacitation involves physiological changes that spermatozoa must undergo in the female reproductive tract or in vitro to obtain the ability to bind, penetrate and fertilize the egg. Up to date, several methods have been developed to characterize this complex biological process. The goal of the presented study is to mutually compare several fluorescent techniques, check their ability to detect changes in molecular processes during the capacitation progress and determine their ability to predict the percentage of acrosome reacted (AR) sperm after the exposure to solubilized zona pellucida (ZP). The capacitation process was analyzed using four fluorescent techniques: 1. chlortetracycline (CTC) staining, 2. anti-acrosin antibody (ACR.2) assay, 3. anti-phosphotyrosine (pY) antibody assay, 4. fluorescein isothiocyanate-conjugated phalloidin (FITC-phall) assay. All these methods were tested using fluorescent microscopy and flow cytometry. Results: All selected methods are capable to detect the capacitation progress of boar sperm in vitro, but there are significant differences in their outcome when using fluorescent microscopy or flow cytometry experimental arrangements and subsequent statistical analysis (KW-ANOVA). Also, the ability to predict the absolute numbers of sperm which will undergo ZP-induced AR differ significantly (CTC and ACR.2 gave the best predictions). Conclusions: Our study compared four largely used methods used to characterize capacitation process, highlighted their differences and showed that all are able to detect capacitation progress, CTC and ACR.2 are furthermore able to accurately predict the percentage of AR sperm after ZP-induced AR.

Male germ cells experience a drastic chromatin remodeling through the nucleo-histone to nucleo-protamine (NH-NP) transition necessary for proper sperm functionality. Post-translational modifications (PTMs) of H4 Lys5, such as acetylation (H4K5ac), play a crucial role in epigenetic control of nucleosome disassembly facilitating protamine incorporation into paternal DNA. It has been shown that butyrylation on the same residue (H4K5bu) participates in temporal regulation of NH-NP transition in mice, delaying the bromodomain testis specific protein (BRDT)-dependent nucleosome disassembly and potentially marking retained nucleosomes. However, no information was available so far on this modification in human sperm. Here, we report a dual behavior of H4K5bu and H4K5ac in human normal spermatogenesis, suggesting a specific role of H4K5bu during spermatid elongation, coexisting with H4K5ac although with different starting points. This pattern is stable under different testicular pathologies, suggesting a highly conserved function of these modifications. Despite a drastic decrease of both PTMs in condensed spermatids, they are retained in ejaculated sperm, with 30% of non-colocalizing nucleosome clusters, which could reflect differential paternal genome retention. Whereas no apparent effect of these PTMs was observed associated with sperm quality, their presence in mature sperm could entail a potential role in the zygote.

Spermatogenesis starts with the onset of puberty within the seminiferous epithelium of the testes. It is a complex process under intricate control of the endocrine system. Physiological regulations by steroid hormones in general and by estrogens in particular are due to their chemical nature prone to be disrupted by exogenous factors acting as endocrine disruptors (EDs). 17α-Ethynylestradiol (EE2) is an environmental pollutant with a confirmed ED activity and a well-known effect on spermatogenesis and chromatin remodeling in haploid germ cells. The aim of our study was to assess possible effects of two doses (2.5ng/ml; 2.5 μg/ml) of EE2 on both histone-to-protamine exchange and epigenetic profiles during spermatogenesis performing a multi/transgenerational study in mice. Our results demonstrated an impaired histone-to-protamine exchange with a significantly higher histone retention in sperm nuclei of exposed animals, when this process was accompanied by the changes of histone post-translational modifications (PTMs) abundancies with a prominent effect on H3K9Ac and partial changes in protamine 1 promoter methylation status. Furthermore, individual changes in molecular phenotypes were partially transmitted to subsequent generations, when no direct trans-generational effect was observed. Finally, the uncovered specific localization of the histone retention in sperm nuclei and their specific PTMs profile after EE2 exposure may indicate an estrogenic effect on sperm motility and early embryonic development via epigenetic mechanisms.

Mammalian sperm cells are not capable of fertilizing an egg immediately after ejaculation; instead, they must gradually acquire the capacity to fertilize while they travel inside the female reproductive tract. Sperm cells are transported by the muscular activity of the myometrium to the utero-tubal junction (UTJ) before entering the oviduct where they undergo this physiological process, termed capacitation. Since the successful emulation of mammalian sperm capacitation in vitro, which led to the development of in vitro fertilization techniques, sperm capacitation and gamete interaction studies have been mostly carried out under in vitro conditions. Sperm cells are typically incubated in vitro for up to several hours at a concentration of more than 1 million cells per milliliter in the capacitation media inside a 37°C incubator with 5% CO2, mimicking the tubal fluid composed of serum albumin, bicarbonate, and Ca2+. The resultant sperm are functionally and molecularly heterogeneous with respect to acrosome reaction, motility, and phosphorylation. By contrast, in vivo sperm capacitation occurs in a time- and space-dependent manner, with limits on the number of capacitating sperm in the oviduct. The small number of sperm at the fertilization site in vivo are highly homogeneous and uniformly capable of fertilization. This discrepancy makes the degree of correlation between the changes observed from in vitro capacitation as a population average and the fertilizing capacity of sperm less clear. To overcome this issue, we used CLARITY tissue clearing to visualize sperm directly inside the female tract in situ and isolated sperm capacitated in vivo from the oviducts of the female mice after timed mating (Ded et al., 2020). Here, we present a step-by-step protocol to collect in vivo capacitated sperm by detailing a microdissection technique and subsequent preparation steps for fluorescent imaging. The advantage of the microdissection technique over in vitro capacitation is the ability to collect physiologically segregated, homogeneous sperm populations at different stages of capacitation. Compared to CLARITY, this technique is more straightforward and compatible with a broader spectrum of antibodies for downstream imaging studies, as it allows the researcher to avoid a potentially high background from non-sperm cells in the tissue. The disadvantage of this technique is the potential contamination of the isolated sperm from different regions of the oviduct and disruption of the fine molecular structures (e.g., CatSper nanodomains) during sperm isolation, especially when the preparation is not performed swiftly. Hence, we suggest that the combination of both in situ and ex vivo isolated sperm imaging is the best way how to address the molecular features of in vivo capacitated sperm., [摘要]哺乳动物的精子细胞在射精后不能立即使卵子受精；相反，它们必须在进入女性生殖道时逐渐获得受精能力。精子细胞在进入输卵管之前通过子宫肌层的肌肉活动运输到子宫 - 输卵管连接处 (UTJ)，在那里它们经历称为获能的生理过程。自从哺乳动物体外精子获能的成功模拟导致体外受精技术的发展以来，精子获能和配子相互作用研究大多在体外条件下进行。精子细胞通常在体外培养 以每毫升超过 100 万个细胞的浓度在 37℃含有 5% CO 2 的培养箱，模拟由血清白蛋白、碳酸氢盐和 Ca 2+组成的输卵管液。由此产生的精子在顶体反应、运动和磷酸化方面在功能和分子上是异质的。相比之下，体内精子获能以时间和空间依赖的方式发生，输卵管中获能精子的数量有限。体内受精部位的精子数量少，同质性高，能均匀受精。这种差异使得从体外获能观察到的作为群体平均值的变化与精子受精能力之间的相关程度不太清楚。为了克服这个问题，我们使用 CLARITY 组织清除直接在原位显示雌性管道内的精子，并在定时交配后从雌性小鼠的输卵管中分离出体内获能的精子（Ded等人，2020 年）。在这里，我们提出了一个循序渐进的协议，通过详细介绍显微解剖技术和荧光成像的后续准备步骤来收集体内有能力的精子。显微切割技术优于体外获能的优势在于能够在不同的获能阶段收集生理上分离的、同质的精子群。与 CLARITY 相比，该技术更直接，并且与用于下游成像研究的更广泛的抗体兼容，因为它允许研究人员避免来自组织中非精子细胞的潜在高背景。这种技术的缺点是在精子分离过程中，来自输卵管不同区域的分离精子的潜在污染和精细分子结构（例如，CatSper 纳米域）的破坏，特别是当制备不迅速进行时。因此，我们建议结合原位和离体精子成像是解决体内获能精子分子特征的最佳方法。 [背景]精子获能的发现发生在 1950 年代，当时研究人员未能通过将精子直接添加到卵母细胞而未在接近输卵管液的类似成分的培养基中进行任何事先培养而使卵子在体外受精（Austin，1951；Chang，1951） . 从那时起，绝大多数关于获能的生理和分子过程的现有信息都是使用体外系统获得的，这些系统在 1970 年代首次用于通过体外受精（IVF）受孕（Steptoe 和 Edwards , 1976)。如今，发达国家高达 10% 的儿童是通过辅助生殖技术受孕的，例如体外受精和卵胞浆内单精子注射 (ICSI)。随着这一发展，更需要获得有关自然获能过程的详细知识，并了解其与体外获能的区别。该协议使研究人员能够通过从时间交配的雌性小鼠的生殖道 (UTJ、峡部和壶腹) 的不同位置分离精子来研究精子在获能过程的不同阶段的分子特征。该协议也可用于从小鼠身上回收新鲜射出的精子 (Li et al ., 2015; Chung et al .,...