All animals were cared in accordance with the Guiding Principles in the Care and Use of Animals of the US National Institute of Health. All procedures were approved by the Institutional Animal Care and Use Committee of the School of Medical Science, Universidad Nacional de Cuyo (Protocol N° 169/2019).

All chemicals and reagents, unless stated otherwise, were purchased from Sigma-Aldrich Chemical Inc. (St. Louis, United States).

Germinal Vesicle (GV, immature) and Metaphase II (MII, mature) oocytes were obtained from hormonally stimulated CF-1 female mice (8–12 weeks) and bred under controlled conditions of light and temperature. To obtain GV oocytes, females were injected intraperitoneally (i.p.) with 10 IU of purified equine chorionic gonadotropin hormone (PMSG) (Syntex, Argentina), and after 45–48 h cumulus oocyte-cell complexes (COC) were obtained by ovarian puncture. Earle’s balanced salt solution with 0.01% PVA, 0.001% Gentamicin, and 25 mM HEPES buffer, pH 7.3 (MEM/HEPES) was used as the collection medium, supplemented with 2.5 μM Milrinone to inhibit oocyte maturation. To denude the GV oocytes, cumulus cells were mechanically removed using fine-bore pipettes. MII oocytes were obtained from female mice stimulated with 10 IU (i.p.) of PMSG (Syntex, Argentina), followed by 10 IU (i.p.) of human chorionic gonadotropin hCG (Syntex, Argentina) 48 h later. After 13–17 h, the MII oocytes were obtained from the oviductal ampullae, denuded from cumulus cells by brief incubation in hyaluronidase (0.04%) and placed in CZB medium (81.62 mM NaCl, 4.83 mM KCl, 1.70 mM CaCl₂.2H₂O, 1.18 mM MgSO₄.7H₂O, 1.18 mM KH₂PO₄, 0.11 mM EDTA.2Na, 25.12 mM NaHCO₃, 52 mM sodium lactate, 0.27 mM sodium pyruvate, 7 mM taurine, 1 mM L-glutamine, 10 mg/mL gentamicin, supplemented with 0.3% BSA) and covered with mineral oil in a humidified chamber (37°C, 5% CO₂) for the shortest time possible until their use.

The CPP used in this study possesses the following sequence: NH₂-K-(5-FAM)RRRRRRRRRC-CONH₂ (MW 2012,32 Da). It was custom-designed by our research team and was commercially synthesized by Innovagen AB using solid-phase synthesis, achieving a purity grade exceeding 98%. This CPP includes an amide group at the C-terminus of the cysteine to prevent undesired binding, a lysine residue at the N-terminus to provide a primary amino group suitable for protein conjugation, and a polyR sequence that confers a membrane-penetrating capability. Furthermore, 5-FAM(5-carboxyfluorescein) is conjugated covalently to CPP through the lysine residue, allowing the peptide to be visualized using fluorescence microscopy.

After collection, GV and MII oocytes were incubated in CPP in CZB medium. To check the effect of the zona pellucida (ZP) in the CPP internalization, some GV and MII oocytes were subjected to a brief incubation in acid Tyrode to remove the ZP, prior to carrying out the corresponding treatments with CPP. If not, oocytes were incubated with ZP at different concentrations of FAM-CPP: 1, 2 and 3 µM (only for MII oocytes), and 4, 10 and 16 µM (for GV and MII oocytes); at different times: 15, 30, and 60 min; and different temperatures: 37°C in a humidified atmosphere of CO₂ (5%) or 4°C in a refrigerator. Likewise, as a control, a group of GV and MII oocytes was treated with CZB medium without CPP. Following, the ZP was removed by brief incubation in acid Tyrode (pH 2.2) and the oocytes were fixed in paraformaldehyde (3.7%) in Dulbecco’s PBS (DPBS) for 1 h at room temperature (RT). Cells were then washed in blocking solution (BS) containing 3 mg/mL BSA, 100 mM glycine, and 0.01% Tween 20 in DPBS, and mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) following two types of mounting techniques: in chamber or flattened (Figure 1A). Chamber mounting consists of placing two lines of solid vaseline between the slide and the coverslip to create a space where the oocytes are contained with a minimal compression to keep their spherical shape. On the contrary, in the flattened mounting, the lines of solid vaseline are absent and the cells are compressed between the slide and coverslip. Then, samples were sealed and stored at 4°C until viewing.

To inhibit endocytosis, oocytes were incubated in CZB medium containing either 80 µM Dyngo-4a (Abcam) or 20 µM Cytochalasin B (Abcam) for 90 min at 37°C in a humidified atmosphere of 5% CO₂. After washing, oocytes were incubated during 30 min in 10 µM FAM-CPP in the same conditions. Then, ZP was removed, and oocytes were fixed as described in section 2.5. Cells were separated in two groups. One cells group was flatten mounted to analyze the cytoplasmic FAM-CPP fluorescence. The other cells group was permeabilized with Triton X-100 for 15 min, stained with Rhodamine-Phalloidin 555 (1:1000) for 60 min and mounted in chamber. For DNA detection, Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA) containing 1.5 μg/mL Hoechst 33342 (Molecular Probes, Invitrogen) was used. After sealing, slides were stored at 4°C until visualization.

MII oocytes with ZP were incubated with 10 µM FAM-CPP in CZB for 60 min, at 37 °C in a humidified atmosphere of CO₂ (5%). Likewise, as a control, a group of MII oocytes was treated with 5-FAM (without CPP) in CZB. Next, MII oocytes were incubated for 30 min in a solution of 2 µM Tetramethylrhodamine perchlorate ethyl ester (TMRE), which allowed the staining of the mitochondrial membranes to determine their potential. Then, cells were washed twice in MEM/HEPES drops and transferred to a real-time chamber with MEM/HEPES without PVP to fix them on the glass and prevent their movement. In vivo visualization was performed on a laser scanning confocal microscope (FV1000, Olympus) using a UPLSAPO 20X/NA:0.75 objective lens, a pixel resolution of ×512512, and the pinhole fully open (800) to obtain the total fluorescence of the cells. Equatorial images of oocytes were captured using the same microscope setup. The absolute total fluorescence intensity of the cell was quantified using ImageJ software (version 1.42L; NIH, MD).

CG staining was performed as previously described (Paola et al., 2015). Briefly, after MII oocytes incubation in CPP (10 µM), at 37°C, and different times (15, 30, 60 min), ZP was removed by brief incubation in acid Tyrode and oocytes were fixed in paraformaldehyde for 1 h at RT. Then, oocytes were washed in BS, and permeabilized with Triton X-100 for 15 min. After 3 washes in BS, cells were incubated in 25 μg/mL Rhodamine-labeled Lens Culinaris Agglutinin (LCA) in BS for 30 min, after which they were washed again in BS. Cells were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) between slide and coverslip (flattened mounting), sealed, and stored at 4°C until viewing.

The TeTx light chain purification was performed as described by Mayorga et al., 2019 (Mayorga et al., 2020). The plasmid DNA encoding His6-tetanus toxin light chain in pQE3-Qiagen (previously provided by T. Binz from Medizinische Hochschule Hannover, Hannover, Germany) was transformed into E. coli XL-1Blue (Stratagene, La Jolla, CA, United States), and protein expression was induced overnight at 20°C with 0.2 mM IPTG. Purification of recombinant His₆-tetanus toxin (TeNT) was accomplished according to the QIAexpressionist (www.qiagen.com). The CPP peptide (RRRQRRKRRRRQ) and the recombinant toxin (His6-TeNT) were cross-linked by Trilinks Biotechnology under the manufacturing protocol. The primary amines of both toxin and peptide were linked to succinimidyl-4-formyl benzamide (S-4FB) and succinimidyl-6-hydrazino-nicotinamide (SHyNic), respectively. The modified proteins and peptides were then conjugated by a specific chemical reaction between HyNic and 4FB in a particular molar ratio (3:1 M equivalent) at RT by 3 h incubation with aniline 100 mM as a catalyst. After the conjugation reaction, the cross-linkers reagents surplus was removed and the CPP-TeTx was purified on a Sephadex G25 column (MP Biomedicals) in a medium suitable for oocyte incubation (MEM/HEPES). The purity was confirmed by SDS-PAGE analysis.

Incubation of MII oocytes was performed in CPP-TeTx (4 μM) for 15, 30 and 60 min, at 37°C on a thermal stage. After that, ZP was removed by brief incubation in acid Tyrode and the oocytes were fixed in paraformaldehyde for 1 h at RT. Cells were then washed in BS, and permeabilized with Triton X-100 for 15 min. After 3 washes in BS, incubation was performed in Mouse Anti-6X His (Abcam) primary antibody (1:20) for 1 h at RT. After 3 washes in BS, oocytes were incubated for 1 h in DyLight 488 donkey anti-mouse secondary antibody (3 ng/μl, Jackson ImmunoReasearch). After 3 washes, cells were flat mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) on a slide, sealed, and stored at 4°C until visualization.

After MII oocytes incubation in 10 μM FAM-CPP or 4 μM pTxTe for 60 min, at 37°C, the cortical reaction was induced by parthenogenetic activation with SrCl₂ (30 mM) in calcium and magnesium free CZB (85.35 mM NaCl, 4.83 mM KCl, 1.18 mM KH₂PO4, 110 μM EDTA.2Na, 12 mM NaHCO₃ 25, 270 μM Na pyruvate, 52 mM Na lactate, supplemented with 0.001% Gentamicin, 0.01% PVA, 1 mM Glutamine), for 1 h at 37°C in a humidified CO₂ (5%) atmosphere. Then, ZP was removed by brief incubation in acid Tyrode and oocytes were fixed in paraformaldehyde for 1 h at RT. Cells were washed in BS, and permeabilized with Triton X-100 for 15 min. After 3 washes in BS, cells were incubated in 25 μg/mL Rhodamine-labeled Lens Culinaris Agglutinin (LCA) in BS for 30 min, after which they were washed again in BS. Cells were flattened-mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) on a slide, sealed, and stored at 4°C. To demonstrate the specificity of the effect of pTxTe, a group of oocytes was incubated with N,N,N′,N′-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a zinc chelator permeable to cells with a high affinity for zinc. Oocytes were treated with 10 μM TPEN during 15 min at 37°C prior activation with SrCl₂.

The images were acquired with a confocal laser scanning microscope (FV1000, Olympus), using a PLAPON 60x/NA1.42 oil immersion objective lens, at a pixel resolution of ×512 512. The cell images were taken at flat optical fields of the equatorial zone (to analyze either the fluorescence of CPP or the immunolabeled CPP-TeTx) or at the cortical zone (to analyze cortical granule density). The confocal acquisition parameters were constant for all images captured. Using the computer-assisted image quantification software ImageJ (version 1.42L; NIH, MD), the absolute fluorescence intensity of oocytes treated with CPP or CPP-TeTx was quantified by drawing a circumference that took the cytoplasm of the cell, but not the cortical zone, and whose diameter was maintained for all the cells analyzed (Figure 1B). To quantify the density of CG through ImageJ, images were deconvoluted to obtain greater sharpness. The mean obtained from the counting of CG present in four non overlapping equal areas from the oocyte cortex, was used to determine CG density per 100 μm² (CG/100 μm²) for each cell.

Experiments were repeated at least three times. The number of oocytes used for each experiment is indicated in the figure or in legends. Data analysis was performed with GraphPad Prism 8 software. Depending on data distribution (whether parametric or not), statistical significance was determined by Student/Mann Whitney t-test or One-way Analysis of Variance (ANOVA)/Brown-Forsythe and Welch ANOVA/Kruskal–Wallis tests followed by post hoc test for multiple comparisons. Data are expressed as mean ± SEM and p < 0.05 is considered statistically significant.

In this study, we investigated the internalization ability of cell-penetrating peptides across the zona pellucida and plasma membrane of mouse oocytes. As a representative CPP model, we selected KR₉C, a peptide comprising nine consecutive arginine residues. Previous research has indicated that oligoarginine sequences consisting of eight to twelve consecutive residues demonstrate optimal internalization capability for small molecules in mammalian cells (Mitchell et al., 2000; Mueller et al., 2008; Tünnemann et al., 2008). To observe the intracellular behavior of the CPP using fluorescence microscopy, we introduced the 5-FAM (5-carboxyfluorescein) into the peptide sequence. Specifically, FAM was conjugated covalently to the lysine residue (see Materials and Methods), resulting in the peptide sequence K(5-FAM)R₉C, which will be referred to as FAM-CPP hereinafter.

To evaluate if this FAM-CPP was able to cross the oocyte’s plasma membrane, we analyzed the fluorescence intensity in Metaphase II (MII or mature) oocytes after cells incubation at 37 °C in the presence (or absence) of 10 µM CPP. For these assays, MII oocytes with zona pellucida were used.

After fixation, cells were mounted in two different manners: in “chamber” or “flattened” between slide and coverslip (see schematic representation of mountings in Figure 1A). To detect exclusively the cytoplasmic fluorescence, only equatorial zone of the cells was imaged. Confocal microscopy images of oocyte’s equatorial zone showed that FAM-CPP accumulated on the outside of cells and in the polar body; thus, we only analyzed the fluorescence intensity of the equatorial region within the red circle as indicated in the scheme of Figure 1B. The analysis of fluorescence intensity showed that the flattened method was the best mounting manner to obtain a significant signal compared to control cells (Figure 1C). FAM-CPP showed a diffuse pattern of distribution in oocyte cytoplasm, while control cells incubated only with FAM (without CPP) did not shown fluorescence. These results indicated that the fluorescent CPP was able to cross the plasma membrane of mature oocytes with zona pellucida and that the flattened method was the best cell mounting to analyze the FAM-CPP internalization in the assayed conditions.

One of the fundamental conditions for a CPP to be viable as a biotechnological tool is that it has low or no cytotoxicity. Although there are studies with cationic peptides (such as the polyarginine used in this work) that indicate that they have low toxicity and are tolerated by cells at high concentrations (Dinca et al., 2016), we first analyzed the viability of mature oocytes incubated with CPP by determining the mitochondrial membrane potential (MMP). As shown in Figure 2A, after 1 h incubation at 37°C in presence of 10 µM CPP, mature oocytes were alive, indicating that the FAM-CPP was not toxic for the cells. Then, to rule out that that FAM-CPP was entering by endocytosis, we preincubated oocytes with two different endocytosis inhibitors before CPP incubation: Dyngo-4a and Cytochalasin B. Dyngo-4a is a dynamin inhibitor that inhibit clathrin-mediated endocytosis and perturb the actin dynamic (Ferguson and De Camilli, 2012; McCluskey et al., 2013). Cytochalasin B is a known reagent that disassembly F-actin cytoskeleton (Cooper, 1987). Mouse oocytes were preincubated in presence of the endocytosis inhibitors prior FAM-CPP incubation. After fixation, oocytes were either flatten mounted to analyze the cytoplasmic FAM-CPP fluorescence or permeabilized to stain F-actin (see details in Materials and Methods) As shown in Figure 2B, both Dyngo-4a and Cytochalasin B disrupted cortical F-actin when compared to control condition. However, FAM-CPP was observed in the cytoplasm indicating that, even when endocytosis was inhibited, the CPP was internalized. These results suggest that the translocation of FAM-CPP take place by a direct penetration mechanism.

The zona pellucida (ZP) is a glycoprotein matrix that functions as a protective barrier, filtering substances from the extracellular space (Gupta et al., 2012). Because this barrier must be crossed to introduce substances into the oocytes, we analyzed the effect of the presence/absence of ZP on the FAM-CPP internalization. For this assay, the ZP was removed before incubating with the fluorescent CPP as explained in Materials and Methods. As shown in Figure 3, both GV oocytes and MII eggs without ZP were able to incorporate significatively more FAM-CPP than cells with ZP, indicating that ZP hinders the fluorescent CPP incorporation. Nevertheless, for the following assays, we decided to use oocytes with ZP because they are easier to manipulate.

Taken together, these results showed that the FAM-CPP can cross both zona pellucida and plasma membrane of MII oocytes (with and without ZP) and does not affect cell viability. Nevertheless, it is worthy to mention that, even if the peptide can cross de ZP, the internalization is significatively reduced in oocytes with ZP.

There is evidence in the literature on the differential influence of concentration, time and temperature on the internalization of CPP (Fretz et al., 2007; Madani et al., 2011; Copolovici et al., 2014). However, often the mentioned factors act in conjunction with other factors such as the nature of the CPP and the type of cell used. For this reason, we tested how those parameters -concentration, time, and temperature-participate in the internalization of FAM-CPP in two different stages of mouse oocyte maturation: Germinal Vesicle (GV, immature) and Metaphase II (MII, mature) oocytes.

First, we analyzed the concentration effect on the FAM-CPP internalization in GV and MII mouse oocytes. Different concentrations (1–16 µM) were tested in immature and mature oocytes with ZP. Again, cells were imaged at equatorial region inside the red circle as explained previously. As shown in Figures 4A, B, the increasing of fluorescence intensity was directly proportional to FAM-CPP concentration in GV oocytes. CPP was homogeneously distributed on the entire cytoplasm and reached the nucleus (or Germinal Vesicle). In other words, FAM-CPP internalization was concentration-dependent in immature oocytes. When the concentration effect on the FAM-CPP internalization was analyzed in MII oocytes, the fluorescence intensity reached a statistically significant plateau between 4 μM and 10 µM and diminished significantly at 16 µM (Figures 5A, B). These results indicate that, similarly to observed in GV oocytes, FAM-CPP internalization was concentration-dependent in MII oocytes until 4 uM, however it reached a plateau at higher concentrations.

Then, we analyzed the effect of time and temperature simultaneously on FAM-CPP internalization for each maturation stages. GV oocytes were collected and incubated in CZB medium with 10 µM FAM-CPP for 15, 30, and 60 min at 4°C or 37°C. As shown in Figure 6, the FAM-CPP internalization occurred at both temperatures and reached a statistically significant plateau at 15 min. Nevertheless, the FAM-CPP internalization at 4°C was statistically lower than 37°C (Figures 6A, B). Similar assays were performed using MII oocytes. Cells were collected and incubated in CZB medium with 10 µM FAM-CPP for 15, 30, and 60 min at 4°C or 37°C. As shown in Figure 7, similar results were obtained when MII oocytes were incubated at 37°C: the intensity of CPP fluorescence reached a plateau at 15 min. Nevertheless, when the FAM-CPP incubation took place at 4°C, the FAM-CPP internalization was dependent on the incubation time (Figures 7A, B).

Altogether, these results demonstrate that the FAM-CPP internalization in mouse oocytes depends on concentration, time, temperature, and the oocyte maturation stages.

Cortical granules (CG) are organelles localized in the cortical region of mature mouse oocytes and are involved in the cortical reaction. This process is an exocytotic event in which CG fuse with the oocyte plasma membrane after the fusion of the first spermatozoa. Thus, the secretion of CG content avoids the fusion of a second male gamete ensuring the embryo development. Considering that this work aimed to analyze the use of CPP to study cortical reaction in mature mouse oocytes, the next step was to investigate if the FAM-CPP internalization does perturb cortical granules density. Then, cortical granules density was evaluated for different incubation times in mature oocytes. For control and experimental conditions, cells were incubated with FAM and FAM-CPP, respectively. The results showed that, compared to control, the FAM-CPP incubation did not alter the cortical granule density at any assayed time in mature oocytes (Figure 8).

Cortical reaction is a calcium regulated exocytosis, in which cortical granules fuse with the oocyte plasma membrane after mouse oocyte activation. This fusion is mediated by SNARE proteins, and we have proposed a working model for cortical granules exocytosis in mouse oocytes (Paola et al., 2021). The SNARE proteins are proteolyzed by the light chains of the clostridial neurotoxins–tetanus toxin and botulinum toxin (Humeau et al., 2000; Schiavo et al., 2000). Tetanus toxin has two polypeptide chains: the heavy and the light chain. The heavy chain mediates binding, internalization, and translocation of the light chain to the cytosol, and the light chain inhibits exocytosis by cleaving VAMP1, VAMP2 or VAMP3 (Dong et al., 2019). In addition, the catalytic activity of the light chain is zinc-dependent and is used as a tool for the study of exocytosis in different secretory cells (Ahnert-Hilger et al., 1989; Bittner et al., 1989). Previously we have demonstrated that VAMP1 and VAMP3 are expressed in mouse oocytes (Paola et al., 2021). Considering that FAM-CPP internalization is innocuous for mouse oocytes (Figure 2A) and does not perturb cortical granules localization (Figure 8), we hypothesized that a permeable tetanus Toxin light chain (CPP-TeTx) might inhibit cortical reaction by cleaving the VAMPs isoforms expressed in mouse oocytes.

First, we purified the CPP-TeTx as a His6-tagged protein as described in Materials and Methods. We tested if this permeable toxin could internalize into mouse mature oocytes. Because CPP-TeTx is not fluorescent, we verified the protein internalization by immunofluorescence assays using a His6 antibody. As shown in Figure 9, CPP-TeTx was detected into mature oocytes incubated by 15, 30 and 60 min at 37°C (Figure 9A). In this case, two controls were performed: one, to determine the unspecific signal from the secondary antibody (0 min-, incubated only with secondary antibody), and the other, to determine the unspecific signal from histidines present in mature oocytes (0 min+, incubated with primary and secondary antibody). Figure 9B shows that the fluorescence intensity for all analyzed times was statistically higher than 0 min + control and time independent. These results are similar to that obtained with FAM-CPP (see Figure 7) and indicate that CPP-TeTx was internalized into mouse mature oocytes.

Second, to test the functionality of CPP-TeTx during parthenogenetically activated cortical reaction, we analyzed the function of CPP-TeTx using our functional assay. This functional assay consists of quantifying and comparing the cortical granules density in control oocytes (without activator) and in strontium-activated oocytes (positive control, with parthenogenetic activator). The difference between these two conditions is a functional measurement of cortical granule exocytosis (Supplementary Figure S1). Considering that the sequence of permeable peptide is similar for both FAM-CPP and CPP-TeTx, we tested if FAM-CPP internalization may affect the cortical reaction. As shown in Supplementary Figure S1, FAM-CPP did not perturb the cortical reaction activated by strontium chloride. Then, if prior strontium activation an inhibitor is present (CPP-TeTx), cortical granules exocytosis will be inhibited, and cortical granules density will be significantly higher than positive control. Mature oocytes with ZP were incubated in the presence of 4 µM CPP-TeTx at 37 °C during 1 h. Similarly to FAM-CPP (see Figure 5), 4 uM CPP-TeTx was the minimum concentration to reach the internalization plateau. Then, cortical reaction was activated by strontium chloride. Before fixation, ZP of the treated oocytes was removed and cortical granules were stained with Rhodamine-Lens Culinaris Agglutinin (LCA) to evaluate cortical granules density as a measure of cortical reaction (see Materials and Methods for details). As shown in Figure 10, CPP-TeTx significantly inhibited the cortical reaction activated by SrCl₂. CPP-TeTx inhibited significantly cortical reaction by about 50%, suggesting that other tetanus toxin-insensitive protein might be involved in this secretory process. Next, considering that the catalytic activity of the light chain of tetanus Toxin is zinc-dependent, we analyzed the effect of zinc chelation to demonstrate the specificity of CPP-TeTx. We used N,N,N′,N′- Tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), a cell-permeable zinc chelator with a high affinity for zinc. Incubation of CPP-TeTx treated oocytes in 10 μM TPEN prevented the inhibition of cortical granule exocytosis, confirming the specificity of the effect of CPP-TeTx (Figure 10). TPEN incubation alone did not alter the cortical reaction in presence or absence of SrCl₂ (Figure 10).

Altogether, these results confirmed our hypothesis and demonstrated that the permeable tetanus toxin inhibits cortical granule exocytosis in mouse oocytes.