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. 2026 May 25;7(2):RAF250199. doi: 10.1530/RAF-25-0199

JAGGED1 modulates bull spermatozoa acrosome reaction and fertilization

Patrícia Diniz 1,2, Inês Leites 1,2, Rosa M L Neto Pereira 1,2,3, Luísa Mateus 1,2, Luís Lopes-da-Costa 1,2, Elisabete Silva 1,2,✉
PMCID: PMC13202239  PMID: 42148704

Abstract

Graphical Abstract

graphic file with name RAF-25-0199inf1.jpg

Abstract

To acquire fertilizing ability, mammalian spermatozoa (SPZ) must undergo acrosome reaction, which is associated with protein remodeling and relocation. Previous studies showed that Notch ligand JAGGED1 is relocated from the apical to the post-equatorial segment of bull SPZ during acrosome reaction. This study evaluated the effects of JAGGED1 supplementation or functional inhibition on bull SPZ acrosome reaction, and subsequent fertilization and embryo development rates. Swim-up-derived SPZ was treated with JAGGED1 (10, 50, or 100 ng/mL) or anti-JAGGED1 antibody (7.5 μg/mL). Treatment with JAGGED1 reduced the proportion of SPZ with non-reacted acrosome during the spontaneous acrosome reaction (44 vs 10.8% at 50 ng/mL; P < 0.001) and increased the proportion of acrosome-reacted SPZ (24.5 vs 34.6% at 100 ng/mL; P < 0.001) during the induced acrosome reaction with calcium ionophore. Treatment with anti-JAGGED1 antibody lowered the proportion of acrosome-reacted SPZ (39.1 vs 26.1%; P < 0.0001) during the induced acrosome reaction and decreased the fertilization rate (69.7 vs 54.8%; P < 0.0001). Overall, the results indicate that JAGGED1 accelerates the SPZ acrosome reaction, thereby limiting the timespan of fertilization ability. It is hypothesized that JAGGED1 contributes to SPZ cytoskeleton remodeling, interacting with other proteins.

Lay summary

To successfully fertilize an egg, mammalian sperm must go through a series of final changes. These changes involve adjustments and the movement of important proteins within the spermatozoa head. This study examines how a protein called JAGGED1 moves within the bull spermatozoa head as it gets ready to fertilize an egg. When extra protein was added, more sperm were able to complete the final change needed for fertilization. When the sperm’s own JAGGED1 was blocked, fewer completed this change, and fertilization was less successful. Together, these results show that JAGGED1 helps spermatozoa to fertilize an egg and supports successful fertilization.

Keywords: JAGGED1, acrosome reaction, fertilization, spermatozoa, bull

Introduction

Bull infertility and subfertility are responsible for low conception rates in both natural mating and artificial insemination (Butler et al. 2020), resulting in major economic losses in the cattle industry. Similarly, in humans, around 40–50% of the infertility cases are due to a male factor (Kumaresan et al. 2021). Semen analysis has been the primary diagnostic tool for predicting male fertility, based on parameters such as concentration, motility, and morphology. However, the fertility predictive value of this approach remains suboptimal and controversial, as it cannot accurately predict spermatozoa’s fertilizing ability (Tanga et al. 2021). This is well evidenced in about 20% of unexplained subfertility/infertility cases in bulls presenting normal semen parameters (Fair & Lonergan 2018). This may have a significant impact on herd reproductive performance, since semen from a single bull is used for artificial insemination of up to thousands of females (Chenoweth & McPherson 2016). Therefore, research on the molecular mechanisms that regulate the functional competence of SPZ is central to understanding the etiology of defective SPZ and developing clinically valuable fertility biomarkers and therapeutic targets (Panner Selvam et al. 2019). One of the late capacitation-associated events is the SPZ’s ability to undergo acrosome reaction, which is a prerequisite for SPZ-oocyte fusion. The acrosome reaction is a Ca2+-dependent exocytotic event involving the fusion of SPZ outer acrosomal and plasma membranes, resulting in the release of hydrolytic enzymes that degrade the oocyte’s zona pellucida and facilitate SPZ-oocyte fusion (Sun et al. 2011, Yanagimachi 2011). In the past few years, several proteins have been identified in mammalian SPZ that play essential roles in the regulation of acrosome reaction and SPZ-oocyte interactions, such as Ca2+/calmodulin-dependent protein kinase II (Ma et al. 2022, Santos et al. 2025), SPACA (Barbaux et al. 2020, Chen et al. 2023), IZUMO1 (Zafar et al. 2021), and ADAM1/ADAM2 (Wong et al. 2001). Nevertheless, despite its obvious biological and medical relevance, the complex network of proteins that govern these biological processes is far from being fully understood (Nagdas et al. 2016). The relocation of SPZ proteins is one of the key events of the acrosome reaction, and several of these relocated proteins are thereafter involved in the fertilization process (Sebkova et al. 2014). Recently, the group showed that NOTCH proteins are present in bull SPZ and are relocated during the acrosome reaction (Diniz et al. 2024). As the acrosome reaction progresses, NOTCH2 and JAGGED1 move from the head apical region toward the post-equatorial segment, while DLL4 is absent in acrosome-reacted sperm (Diniz et al. 2024). This dynamic redistribution suggests that NOTCH2 and JAGGED1 are linked to acrosome reaction onset, whereas DLL4 is involved in acrosome stabilization. This study aimed to investigate the effect of JAGGED1 supplementation or blockade on bull SPZ acrosome reaction and to evaluate its impact on fertilization and early embryonic development. Based on the previous work, the hypothesis to be tested is that JAGGED1 abundance will modulate the acrosome reaction and its blockade will negatively impact the fertilizing ability and, potentially, the subsequent embryonic developmental competence.

Materials and methods

Effect of JAGGED1 treatment on SPZ viability

As no data on the effect of JAGGED1 on SPZ viability were available, the first step was to test its effects following JAGGED1 supplementation. The effect of JAGGED1 treatment (recombinant human Jagged1; R&D Systems, USA; 1277-JG-050; 96% identity with bovine JAGGED1) on SPZ viability was assessed using frozen-thawed semen from three bulls with previously proven in vitro and in vivo fertility. Motile SPZ population was obtained by the swim-up procedure. Briefly, cryopreserved semen (pool of three straws/bull) was thawed at 37°C for 20 s and layered below a Sperm-TALP medium (BSA-free, Tyrode-albumin-lactate-pyruvate; Sigma, T2397, GmbH, Germany) supplemented with 25 mM NaHCO3, 72.72 mM pyruvic acid (Sigma, P3662), and 0.05 mg/mL gentamycin (Sigma; G1522). Samples were incubated for 1 h at 39°C in a humidified atmosphere containing 5% CO2. After incubation, the upper two-thirds of the medium were recovered and divided into four tubes (1 × 106 SPZ/mL), with one tube allocated to each experimental group. Samples were centrifuged at 200 g for 10 min, and the pellet was resuspended in Sperm-TALP supplemented with 60 μg/mL heparin, PHE (10 mM penicillamine, 20 mM hypotaurine, and 0.25 mM epinephrine), 0.05 mg/mL gentamycin (capacitation medium) and with JAGGED1 (10, 50, or 100 ng/mL) or left untreated (control). Motility was evaluated subjectively, and samples were incubated for 1 h at 39°C under 5% CO2 to allow capacitation. Viability was then assessed using the Live/Dead Viability Kit (Invitrogen, Thermo Fisher Scientific, USA; L-7011), according to the manufacturer’s instructions. Images were acquired with an inverted epifluorescence microscope (Leica DMR, GmbH, Germany). Three samples per treatment and five fields per sample were analyzed, evaluating approximately 350–400 SPZ per group based on green (live SPZ) and red (dead SPZ) fluorescence.

Effect of JAGGED1 treatment on SPZ acrosome reaction

The effects of JAGGED1 treatment were evaluated in non-capacitated SPZ and during SPZ capacitation, under both spontaneous and induced acrosome reaction (Fig. 1A). To assess the treatment under non-capacitation conditions and during the spontaneous acrosome reaction, frozen-thawed bovine SPZ from three bulls were incubated with JAGGED1 (10 or 50 ng/mL) during the swim-up procedure (1 h). For samples evaluated under capacitation conditions, JAGGED1 treatment was performed for 90 min or 3 h in the capacitation medium (Fig. 1A). Briefly, motile frozen-thawed SPZ (1 × 107 SPZ/mL) obtained following swim-up were incubated in the capacitation medium, as described above, and supplemented with 10, 50, or 100 ng/mL of JAGGED1. Thereafter, samples were centrifuged at 200 g for 10 min at room temperature (RT) and washed twice with PBS (phosphate-buffered saline). Samples were then treated with 0.7 μM (Yoshida et al. 2010) of calcium ionophore (CaI; Sigma, A23187) (45 min at 39°C in a 5% CO2 humidified atmosphere), to induce the acrosome reaction, or left untreated to evaluate the spontaneous acrosome reaction. Control samples, in which JAGGED1 was replaced by the corresponding IgG (Recombinant Human IgG1; 110-HG-100; R&D Systems) at a concentration of 100 ng/mL, were run in parallel. Spermatozoa were then processed for fluorescence to allow the differentiation of the acrosome status: NR – non-reacted, R – reacting, AR – acrosome reacted/intact (Supplementary Fig. S1 (see section on Supplementary materials given at the end of the article)) (Yoshida et al. 2010). For each treatment, samples from three bulls (pool of three straws/bull) were evaluated, and a minimum of 300 SPZ in five fields per sample were analyzed for acrosome status classification.

Figure 1.

Figure 1

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Experimental design of the JAGGED1 supplementation and in vitro fertilization assays.

Effect of anti-JAGGED1 neutralizing antibody treatment on SPZ acrosome reaction

These experiments were conducted using the anti-JAGGED1 neutralizing antibody (goat anti-human Jagged 1 antibody; R&D Systems; AF1277; 96% of identity with bovine JAGGED1). The antibody is directed to the JAGGED1 region that includes the DSL domain and initial EGF-like repeats, which determine the cross talk with receptors/proteins, thus functionally blocking JAGGED1 binding activity. Motile frozen-thawed bovine SPZ obtained by swim-up were capacitated, as described above, and treated with the anti-JAGGED1 neutralizing antibody (7.5 μg/mL) or with the goat IgG isotype control (7.5 μg/mL; Abcam, UK; ab37373) for 1 h. The antibody concentration was selected based on the concentration range recommended in the antibody datasheet and previously reported to effectively neutralize JAGGED1 activity (De Falco et al. 2018). After 1 h of treatment with the neutralizing antibody or the IgG control, samples were centrifuged at 200 g for 10 min. To assess the effect of treatment on the acrosome reaction, samples (2 × 106 SPZ/mL) were then resuspended in 1 mL of BSA-free Sperm-TALP, with or without CaI, and incubated for 45 min at 39°C in a 5% humidified CO2 atmosphere. For each treatment, samples from three bulls (pools of three straws per bull), in three independent assays, were analyzed by fluorescence microscopy, and acrosome status was evaluated in a range of 300–850 SPZ in five fields per sample.

Acrosome staining

Spermatozoa were cytospin-centrifuged for 4 min at 1,500 rpm onto a slide and air-dried for 10 min. The slides were fixed with 1% paraformaldehyde (PFA; Sigma, 8.18715) for 30 min at 4°C, washed twice with PBS for 5 min, and then blocked with 2.5% bovine serum albumin (BSA; Sigma, A7906) for 1 h in a humidified dark chamber at RT. Slides were then washed with PBS 1x and incubated with the peanut agglutinin (3 μg/mL; PNA; lectin PNA Alexa Fluor™ 488, L21409, Invitrogen™) for 15 min to stain acrosomes. This was followed by washing and incubation with Hoechst 33258 (1 μg/mL; Sigma, B2883) for 10 min to counterstain the nuclei, after which slides were washed and mounted with the ProLong™Gold antifade mounting medium (Invitrogen™, P36934). Images were acquired in an inverted epifluorescence microscope (Leica, DMR) and analyzed and treated with Adobe Photoshop (CS5 21.1.1).

In vitro fertilization assays

The in vitro fertilization assays were performed with frozen-thawed semen from two bulls with previously proven in vitro and in vivo fertility. Semen was thawed at 37°C for 20 s and treated during the swim-up procedure with one of the following : i) 50 ng/mL of protein IgG isotype control; ii) 50 ng/mL of JAGGED1; iii) 7.5 μg/mL of antibody IgG control; and iv) 7.5 μg/mL of anti-JAGGED1 neutralizing antibody (Fig. 1B). Bovine ovaries were collected immediately after slaughter at a local abattoir and transported to the laboratory within 2.5 h, at 37°C in Dulbecco’s PBS (Gibco, Thermo Fisher Scientific, UK; 14040-91) supplemented with 0.15% BSA (w/v, Fraction V, Sigma, A-7888) and 0.05 mg/mL kanamycin (Sigma, K-4000). In the laboratory, cumulus oocyte complexes (COCs) were aspirated from follicles with 2–8 mm in diameter, and oocytes with at least three layers of compact cumulus cells and an evenly granulated cytoplasm were washed and selected for in vitro maturation. Briefly, selected COCs were matured in TCM199 with Earle’s salts (Sigma, M2154), supplemented with L-glutamine and 25 mM HEPES (Gibco, 22340-020), 10% of fetal calf serum (FCS; Sigma, F7524), 10 IU/mL of follicle-stimulating hormone (FSH; Sigma, 2293), 100 IU/mL penicillin, and 100 mg/mL of streptomycin (Sigma, P0781), for 22 h in an incubator at 39°C with 5% CO2 in a humidified atmosphere (Fig. 1B). The oocyte maturation status was evaluated by cumulus cell expansion under a stereomicroscope, and only oocytes fully expanded were considered matured and proceeded to fertilization. Briefly, COCs were fertilized on Day 0 (insemination day) in a modified Tyrode’s medium supplemented with 25 mM NaHCO3, 5.94 IU/mL heparin (Sigma, H-3393), and PHE. Groups of 20–25 COCs were co-incubated with treated SPZ (1 × 106 SPZ/mL). On Day 2 post-insemination, the cumulus cells were removed by vortexing for 3 min, and the cleaved embryos were selected and transferred to 100 μL droplets of synthetic oviduct fluid containing amino acids (SOFaa; supplementary Table S1). The embryos were in vitro cultured under mineral oil at 39°C in 5% CO2. The cleavage rate was assessed on Day 2, and blastocyst development was evaluated on Days 7 and 8 (Day 0 = insemination day). The experiment was replicated three times.

Statistical analysis

For the effect of JAGGED1 treatment on SPZ viability, data were analyzed using linear mixed-effects models to assess the effect of treatment on sperm viability. Treatment was included as a fixed effect and bull as a random effect. For the effect of JAGGED1 treatment on the SPZ acrosome reaction, data are presented as the proportion of SPZ exhibiting intact, reacting, or reacted acrosomes, and as the proportion of cleaved embryos and blastocysts. Differences among treatments were analyzed using the chi-square (X2) test to compare categorical distributions of acrosome status or embryo rates. Statistical significance was considered at P < 0.05.

Results

Effect of JAGGED1 supplementation on bull SPZ viability

Sperm motility following swim-up was between 75 and 80%, indicating an efficient recovery of viable, motile SPZ, and their suitability for subsequent analysis. As no differences were observed among bulls, the data were pooled for analysis. As shown in Table 1 and Fig. 2, no differences were found (P > 0.05) in SPZ viability between the control and JAGGED1-treated groups, evidencing the innocuous effect of exogenous JAGGED1 on SPZ viability.

Table 1.

Effect of JAGGED1 supplementation on bull spermatozoa viability. Results are expressed as the % of live spermatozoa (live SPZ/total SPZ).

Group Viability
Control 65.0% (264/406)
JAGGED1 10 68.8% (273/397)
JAGGED1 50 67.7% (247/365)
JAGGED1 100 68.0% (242/356)
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Figure 2.

Figure 2

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Representative images of bull spermatozoa stained with SYBR-14 (green; live SPZ) and propidium iodide (red; dead SPZ) (live/dead kit; Thermo Fisher Scientific), after treatment with JAGGED1 at concentrations of 10, 50, and 100 ng/mL. 400× magnification.

Effect of JAGGED1 supplementation on bull SPZ acrosome reaction

Under non-capacitating conditions and without acrosome reaction induction, JAGGED1 supplementation resulted in a concentration-dependent decrease in non-reacted (NR) SPZ relative to untreated controls (P < 0.001) (Figs 3 and 4). Compared to control (44%), NR proportion was lowered to 31.2% (JAGGED1 10 ng/mL) and 10.8% (JAGGED1 50 ng/mL), and this was accompanied by an increase (P < 0.001) of reacting (R) forms, although the proportion of acrosome-reacted (AR) SPZ was not affected and remained low in all groups. Under capacitation conditions (90 min) with either 10 ng/mL or 100 ng/mL, JAGGED1 increased (P < 0.05) the proportion of AR SPZ in both the spontaneous and the induced acrosome reaction, but this effect was more pronounced in the induced acrosome reaction (Figs 5A, B and 6). In the spontaneous acrosome reaction, JAGGED1 doses of 10 and 100 ng/mL increased the proportion of AR SPZ from 4.8% in the control group to 12.5 and 16.2%, respectively (P < 0.05 and P < 0.001, respectively). In the induced acrosome reaction, the proportion of AR SPZ increased from 24.5% in the control group to 31.8 and 34.6% in the JAGGED1 treatments (10 ng/mL and 100 ng/mL, P < 0.001), whereas the 50 ng/mL dose had a lower effect (30.5%, P < 0.05). To evaluate a treatment time-dependent effect, JAGGED1 supplementation was also tested after 3 h of treatment (Fig. 5C and D). Under the 3-h capacitation condition, JAGGED1 treatment only increased the proportion of AR SPZ in the spontaneous acrosome reaction. Compared to the control group (9.5%), the doses of 10 ng/mL and 100 ng/mL increased AR SPZ proportion to 13.8% (P < 0.001) and 11.1% (P < 0.05), whereas in the induced acrosome reaction, it only decreased the proportion of NR SPZ (10.1 versus 5.6%; P < 0.001) but had no effect on the proportion of AR SPZ.

Figure 3.

Figure 3

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Effect of supplementation with JAGGED1 (10 or 50 ng/mL) under non-capacitation conditions, on the spontaneous acrosome reaction of bull spermatozoa. ***P < 0.001. NR, non-reacted SPZ; R, reacting SPZ; AR, acrosome-reacted SPZ.

Figure 4.

Figure 4

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Representative images of the spontaneous acrosome reaction under non-capacitation conditions in untreated control and JAGGED1-treated (10 and 50 ng/mL) bull spermatozoa. Acrosome staining with Alexa® 488 – PNA (green). Counterstaining with Hoechst 33258 (blue). Epifluorescence analysis, ×400 magnification. Red arrows show SPZ with a reacting acrosome (R); white arrows show SPZ with a non-reacted acrosome (NR). 400× magnification.

Figure 5.

Figure 5

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Effect of JAGGED1 treatment on the spontaneous (A and C) and induced (B and D) acrosome reaction of bull spermatozoa under capacitation conditions after 90 min (A and B) and 3 h (C and D). *P < 0.05; ***P < 0.001.

Figure 6.

Figure 6

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Representative images of the spontaneous and induced acrosome reaction after 90 min of JAGGED1 treatment (10, 50, and 100 ng/mL) of bull spermatozoa. Acrosome staining with Alexa® 488 – PNA (green). Counterstaining with Hoechst 33258 (blue). Epifluorescence analysis, ×400 magnification. White arrows show SPZ with a non-reacted acrosome (NR); red arrows show SPZ with a reacting acrosome (R); yellow arrows show acrosome-reacted SPZ (AR).

Effect of anti-JAGGED1 treatment on acrosome reaction of bull spermatozoa

JAGGED1 neutralizing antibody did not affect the SPZ spontaneous acrosome reaction. In contrast, following the induction of the acrosome reaction, the neutralizing antibody anti-JAGGED1 lowered the proportion of AR SPZ from 39.1% in the control group to 26.1% (P < 0.0001) (Fig. 7).

Figure 7.

Figure 7

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Effect of JAGGED1 neutralizing antibody on acrosome status of bull spermatozoa following the spontaneous (without CaI) and induced acrosome reaction (CaI). *P < 0.0001.

Effect of treatment of bull spermatozoa with JAGGED1 or anti-JAGGED1 antibody on fertilization and embryo development

Stimulation of SPZ with JAGGED1 decreased the fertilization rates (P < 0.01) and did not affect early embryo development (Table 2). Similarly, treatment with anti-JAGGED1 neutralizing antibody decreased (P < 0.001) the in vitro fertilization rate, compared to the control group (54.8 vs 69.7%). However, there was a difference in fertilization and embryo development rates among bulls.

Table 2.

Fertilization and embryo development rates using SPZ treated with JAGGED1 or anti-JAGGED1 antibody.

Group Fertilization Embryo development
Oocytes, n Cleavage, n (%) Blastocysts, n (%)
Control 226 162 (71.2) 27 (17.3)
Protein IgG isotype 212 140 (66.0) 21 (15.0)
JAGGED1 protein 240 131 (54.6)* 21 (16.0)
Antibody IgG isotype 261 182 (69.7) 23 (12.6)
Neutralizing JAGGED1 219 120 (54.8)† 10 (8.3)
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*

P < 0.01.

†

P < 0.001.

Discussion

This study evaluated the presence and role of JAGGED1 in the acrosome reaction, fertilization, and embryo developmental competence of bull SPZ. Through JAGGED1 supplementation and neutralization experiments, results provide evidence that JAGGED1 participates in bovine SPZ acrosome reaction and fertilization events. Previous studies by our group have shown that the ligand JAGGED1 is expressed in bull epididymal tissue and is present in both epididymal and mature ejaculated bovine SPZ (Diniz et al. 2024). JAGGED1 showed a localization pattern linked to the acrosome status, relocating during the spontaneous and induced acrosome reaction, from the apical to the post-equatorial region of the sperm head (Diniz et al. 2024). This dynamic relocation pattern is characteristic of proteins involved in the acrosome reaction, SPZ-oocyte fusion, and fertilization (Frolikova et al. 2016, Inoue & Wada 2018, Barbaux et al. 2020, Girela et al. 2025). JAGGED1 supplementation induced the highest acrosome reaction proportion following the shorter capacitation (90 min), in both the spontaneous and induced acrosome reaction. However, even after the stimulation with calcium ionophore, only a subpopulation of SPZ (∼40%) underwent acrosome reaction. This subpopulation reflects the fraction of SPZ that was capacitated, since only capacitated SPZ can undergo the acrosome reaction. This is consistent with physiological heterogeneity within sperm populations, even within a swim-up selected population, which influences the proportion of SPZ competent for the acrosome reaction (Baro Graf et al. 2020, Aldana et al. 2021). Treatment with JAGGED1 neutralizing antibody impaired the SPZ ability to undergo acrosome reaction in response to calcium ionophore induction, reinforcing the involvement of JAGGED1 in the SPZ acrosome reaction event.

The acrosome reaction is a tightly regulated Ca2+-dependent exocytic event triggered by SPZ-zona pellucida binding, which causes an intracellular SPZ Ca2+ influx, which drives the fusion of the SPZ acrosomal and plasma membranes (Michaut et al. 2000, Suhaiman & Belmonte 2024). Induction of the acrosome reaction with calcium ionophore (A23187), a lipid-soluble compound that delivers Ca2+ across biological membranes, transferring it into the SPZ cytoplasm, bypasses the physiologic receptor-mediated signaling that occurs in the natural event. This indicates that JAGGED1 participates in the acrosome reaction event, downstream of calcium entry. Nevertheless, the precise mechanism may only be hypothesized, requiring additional research. JAGGED1 may interact with other SPZ proteins through a non-canonical Notch signaling pathway, without the involvement of a Notch receptor, a mechanism also described in somatic cells (Kamalakar et al. 2019, Zhou et al. 2022, Kaimari et al. 2023), namely, in the regulation of actin remodeling (Frolikova et al. 2016, Liu et al. 2019), as the acrosome reaction requires a dynamic remodeling of the actin cytoskeleton. In this concept, JAGGED1 may interact with cytoskeletal proteins, where F-actin serves as a structural scaffold for components of the pathway, and depolymerization of its network facilitates the migration of proteins to the equatorial segment (Breitbart et al. 2005, Romarowski et al. 2018, Barbaux et al. 2020), a key feature of the acrosome reaction, observed with JAGGED1. Alternatively, JAGGED1 may canonically interact with a Notch receptor, a Notch cis signaling within the SPZ. This hypothesis may be supported by the similar SPZ localization and relocation patterns of NOTCH2 and JAGGED1 during the bull SPZ acrosome reaction (Diniz et al. 2024). In somatic cells, Notch signaling is a pleiotropic pathway that cross talks with other pathways, through both canonical and non-canonical signaling (LaFoya et al. 2016). Several protein kinases that interact with Notch signaling in other systems, such as CaMKII, PKA, and the PI3K/AKT pathway, are key regulators of the acrosome reaction (Ducibella & Fissore 2008, Rotfeld et al. 2014, Stival et al. 2015, Santos et al. 2025). CaMKII regulates the timing of the acrosome reaction by targeting specific substrates involved in membrane fusion and cytoskeletal remodeling. Once activated, CaMKII phosphorylates cytoskeletal regulators and promotes actin polymerization to stabilize membrane domains and suppress premature acrosome exocytosis (Orta et al. 2018). PKA promotes PI3K activation leading to PIP3 production and subsequent AKT activation, which phosphorylates proteins required for cytoskeletal remodeling and exocytosis (Shorning et al. 2020). Thus, NOTCH2 and JAGGED1 may interact with the above signaling cascades, contributing to cytoskeletal remodeling and facilitating the membrane fusion process.

Results showed that either sperm JAGGED1 supplementation or its inhibition significantly reduced the fertilization rate. This inhibitory effect may result from the observed decrease in the acrosome reaction. In particular, the reduction in the fertilization rate after sperm treatment with JAGGED1 supports their role in accelerating the acrosome reaction, inducing it prematurely, and therefore reducing the number of spermatozoa able to fertilize the oocyte. However, an alternative or simultaneous effect on the sperm–oocyte fusion process may be involved. Indeed, the relocation of JAGGED1, during the acrosome reaction, to the equatorial segment, where sperm–oocyte fusion is initiated, suggests this latter involvement, as demonstrated for other SPZ proteins (Castillo et al. 2019, Barbaux et al. 2020). One may suggest that it acts in a dose-dependent manner, possibly through the interaction with other proteins/receptors in the oocyte.

In conclusion, this study evidences a role for JAGGED1 in the bull SPZ acrosome reaction and fertilization. JAGGED1 supplementation increased the proportion of acrosome-reacted SPZ, whereas the opposite was observed following the functional blocking of JAGGED1. The hypothesis put forward for its role in the acrosome reaction is that JAGGED1 contributes to cytoskeletal remodeling through interaction with other SPZ proteins, by either canonical or non-canonical Notch signaling. Sperm JAGGED1 supplementation or inhibition decreased the fertilization rate, which may be related not only to its effect on acrosome reaction but also to a direct role in fertilization, through interaction with other proteins/receptors in the oocyte. However, further studies are needed to elucidate the precise mechanisms underlying JAGGED1 involvement in the acrosome reaction and fertilization. This could have implications for the identification of male fertility markers and the improvement of assisted reproductive technologies.

Supplementary materials

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the work reported.

Funding

This study was supported by the Portuguese Foundation for Science and Technology (FCT), under projects UID/276/2025 (CIISA) and AL4AnimalS LA/P/0059/2020 (AL4AnimalS). PD was funded by FCT grant SFRH/BD/130536/2017. ES was funded by FCT (https://doi.org/10.54499/CEECINST/00140/2021/CP2807/CT0001). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Authors contribution statement

PD was involved in the investigation, methodology, formal analysis, and writing and preparation of the original draft. IL and LM reviewed and edited the manuscript. RLN conducted the IVF methodology. ES and LLC were involved in the conceptualization, supervision, funding acquisition, methodology and validation, formal analysis, and reviewing and editing of the manuscript. All authors read and approved the final manuscript.

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