Beyond Shedding: Conserved Retention of ZAN After the Acrosome Reaction Across Rabbit, Boar, and Bull Sperm

Miranda Hernández-Falcó; Lucía Díaz-Fuster; Núria Ferrer-Cortes; Laura Robles-Gómez; Paula Sáez-Espinosa; María José Gómez-Torres

doi:10.20944/preprints202607.1337.v1

Submitted:

17 July 2026

Posted:

17 July 2026

You are already at the latest version

Abstract

Zonadhesin (ZAN) is a sperm protein currently known for its role in binding to the oocyte zona pellucida. Although its localization has been described in ejaculated and capacitated spermatozoa from mice, pigs and rabbits, its distribution following the acrosome reaction remains poorly understood. Here, we characterized ZAN localization in rabbit, boar, and bull spermatozoa under in vitro conditions mimicking early fertilization events. Ejaculated spermatozoa, swim-up-selected spermatozoa in capacitation medium, and acrosome reaction-induced spermatozoa were analyzed. Immunofluorescence was performed using an antibody against the MAM domain of ZAN to determine its localization and PNA-FITC to assess acrosomal status, while protein abundance was assessed by quantitative LC-MS/MS. Consistent with previous reports, ZAN was detected in the anterior region of intact spermatozoa. However, the MAM-specific antibody also revealed post-acrosomal localization. Species-specific patterns were observed in intact spermatozoa: rabbit and bull showed acrosomal staining with or without post-acrosomal labeling, whereas boar displayed predominant pre-equatorial and post-acrosomal localization. Although ZAN labeling decreased after acrosomal exocytosis, the protein was consistently retained in the post-acrosomal region. These findings suggest ZAN contributes not only to sperm–ZP binding but also to later fertilization events, including sperm–oolemma interaction and membrane fusion.

Keywords:

acrosomal reaction

;

andrology

;

capacitation

;

fertilization

;

fusion

;

gamete interaction

;

mammals

;

reproduction spermatozoa

;

ZAN (zonadhesin)

Subject:

Biology and Life Sciences - Life Sciences

1. Introduction

During fertilization in mammals, the protein Zonadhesin (ZAN) plays an important role in binding the spermatozoon to the zona pellucida (ZP) of the oocyte. This protein was originally isolated and characterized from porcine sperm as a large protein (~300-350 kDa). In this mosaic protein, there is a MAM-type domains (memprin/A5 antigen/tyrosine phosphatase μ receptor), a mucin-like domain, a von Willebrand factor-homologous D domain, and an epidermal growth factor (EGF) [1]. This protein has been observed in various mammalian species [2], including pigs [1,3,4], mice [5,6], humans [7], rabbits [8], and hamsters [9]. Despite some interspecific differences in the number of D domains, the MAM-type domain is highly conservated, including four preserved cysteine residues and hydrophobic/aromatic residues retained through evolution, which are crucial for maintaining its structure and potential role in adhesion [5].

It is known that each of these domains can play a role in adhesion [6,8], either through interactions between proteins or between proteins and carbohydrates. According to Gao and Garbers (1998) [5], the mucin-like domain of ZAN may prevent non-specific interaction between sperm and oviductal isthmus cells, and that the EGF domain may facilitate binding to the ZP. Consistent with an association between ZAN and the ZP, Lea et al. (2001) detected ZAN in acrosomal remnants attached to the ZP following the acrosome reaction [8]. Although the sperm of Zan-deleted mice were less likely to recognize the ZP, they were not infertile, suggesting redundancy or compensatory mechanisms [6].

Electron microscopy studies in pigs [4] and hamsters [9], using antibodies recognizing the functional form of ZAN, localized the protein predominantly to the outer acrosomal membrane during spermiogenesis. In hamster round spermatids, however, ZAN was also detected at the inner acrosomal membrane before becoming largely restricted to the outer acrosomal membrane in late spermatids. In addition, weak labeling in the equatorial segment has been reported in intact rodent spermatozoa, suggesting species-specific differences in ZAN localization. Nevertheless, ZAN has been reported to be exposed during capacitation and proposed as a marker of this process [10]. To explain these findings, a kiss-and-run mechanism has been proposed, involving transient fusion pores between the plasma and acrosomal membranes that allow surface exposure without complete acrosomal exocytosis.

Despite advances in the study of ZAN, there is still limited information available on its interaction with other proteins during gamete recognition. Nor is it known how its exposure during the acrosome reaction facilitates sperm adhesion and penetration of the ZP. Accurately understanding the molecular mechanisms that regulate the reorganization of membrane proteins on the surface of spermatozoa remains a challenge [11]. Sperm capacitation and fertilization are highly complex processes involving multiple interconnected proteins and biochemical events [12], making it difficult to isolate the specific role of ZAN [10]. Furthermore, its study is affected by variability between species and methodological limitations [11].

To overcome these barriers, additional studies are needed that use techniques such as immunocytochemistry and immunofluorescence in a complementary manner to determine more precisely the distribution of ZAN in spermatozoa. Accordingly, the present study investigates ZAN through a multi-species comparative framework under in vitro conditions mimicking early fertilization events, specifically capacitation and acrosome reaction. Furthermore, complementary analytical approaches were integrated to ensure comprehensive characterization, employing fluorescence microscope to assess the distribution across distinct sperm head domains and proteomics for quantitative comparation.

Overall, our findings demonstrate that ZAN remains associated with mammalian sperm following the acrosome reaction across the species examined, supporting a conserved pattern that points to a potential role during the final steps of fertilization, particularly in sperm membrane interaction with the oolema.

2. Results

2.1. Evaluation of Acrosomal Status

Cells showing green fluorescence in the acrosome were classified as non-reacted, while those with equatorial region labelled or without labeling, were considered reacted (see Figure 1a, Figure 2a and Figure 3a). No significant differences in spontaneous acrosome reaction rates were observed before and after swim-up selection in capacitation medium in rabbit and bull spermatozoa (Rabbit E: 10.23% vs. S: 28.95%; Bull E: 1.83% vs. S: 3.17%), whereas there were significant differences in boar spermatozoa (E: 4.00% vs. S: 22.83% p < 0.01). Induction of the acrosome reaction significantly increased the proportion of acrosome-reacted spermatozoa compared with fresh samples in rabbit, boar, and bull (E vs. AR; p < 0.01).

2.2. Permeabilization Reveals ZAN in Post-Acrosomal Domain of Rabbit Intact Spermatozoa

The analysis of ZAN distribution in rabbit sperm revealed five different staining patterns (Figure 1a): pattern 1 characterized by speckled staining in the apical ridge and pre-equatorial subdomain; pattern 2 exhibited strong apical ridge and pre-equatorial subdomain staining with speckled staining in the post-acrosomal domain; pattern 3 showed staining at the equatorial bulges as well as post-acrosomal domain; pattern 4 displayed staining only at the post-acrosomal domain; and pattern 5 without labelling.

As a result of swim-up selection in capacitation medium, the distribution of ZAN at the equatorial bulges as well as the post-acrosomal domain increased significantly from 0.99% to 17.59% (Pattern 3; Mann–Whitney U test; p = 0.001). Conversely, no significant differences were observed in the remaining patterns (Mann–Whitney U test, p > 0.05) when comparing ejaculated sperm with those selected in capacitation medium: pattern 1 (62.58% vs. 37.03%), pattern 2 (28.53% vs. 30.97%), pattern 4 (8.02% vs. 9.18%), and pattern 5 (2.53% vs. 5.50%; see Figure 1b for details).

2.3. Dynamic Remodeling of ZAN Patterns During Swim-up Selection in Capacitation Medium in Porcine Sperm

Given the characteristic membrane domains organization of porcine sperm cells, ZAN exhibited the identical set of four localization patterns previously described by our group for IZUMO1 protein [13], though their relative abundances were different. It should be noted that the patterns were as follows (Figure 2a): pattern 1 characterized by speckled staining in the pre-equatorial subdomain and post-acrosomal domain, pattern 2 displaying strong apical ridge staining with speckled staining in the pre-equatorial subdomain and post-acrosomal domain, pattern 3 exhibiting speckled staining in the post-acrosomal domain, and pattern 4 without labelling.

Sperm swim-up selection in capacitation medium induced significant changes in the relative distribution among the four localization patterns (p < 0.05 for each pattern; Figure 2b). For the variables meeting the assumption of equal variances, independent samples t-tests showed that spermatozoa displaying strong apical ridge staining with speckled staining in the pre-equatorial subdomain and post-acrosomal domain were significantly higher in ejaculated sperm compared selected in capacitation medium sperm (pattern 2: from 84.54% to 11.63%; Student’s t-tests; p < 0.001); meanwhile those spermatozoa exhibiting speckled staining only in the post-acrosomal domain were higher after selection in capacitation medium (pattern 3: from 1.64% to 14.70%; Student’s t-tests; p < 0.001).

For variables with unequal variances, Welch’s t-tests revealed that spermatozoa with labelling in the pre-equatorial subdomain and post-acrosomal domain were significantly higher after selection in capacitation medium (pattern 1: from 11.46% to 65.54%; Welch’s t-test; p < 0.001), as were in spermatozoa without labelling (pattern 4: from 2.36% to 8.13%; Welch’s t-test; p = 0.038).

2.4. Swim-up Selection in Capacitation Medium Enhances ZAN Protein Localization in the Acrosomal Domain of Bull Sperm

Upon analysis of the ZAN distribution in bull sperm, the following four staining patterns were observed (Figure 3a): pattern 1 characterized by speckled staining in the apical ridge and pre-equatorial subdomain; pattern 2 exhibited strong apical ridge and pre-equatorial subdomain staining with speckled staining in the post-acrosomal domain; pattern 3 displayed staining only at the post-acrosomal domain; and pattern 4 without labelling.

Comparison of ejaculated and swim-up-selected bull sperm in capacitation medium (Figure 3b) revealed a significant difference only in the distribution of pattern 1 characterized by speckled staining in the apical ridge and pre-equatorial subdomain with an increment from 4.21% in E to 19.56% in S (Welch’s t-test; p < 0.001). No significant differences were observed for apical ridge and pre-equatorial subdomain staining with speckled staining in the post-acrosomal domain (pattern 2; E: 55.90% vs. S: 24.38%), or for pattern 3, characterized by staining only at the post-acrosomal domain (E: 17.44% vs. S:11.04%), as assessed by Student’s t-tests (p > 0.05). Similarly, the absence of labelling (pattern 4), did not differ significantly between groups (E: 22.44% vs. S: 23.45%; Welch’s t-test; p > 0.05).

2.5. Conservation of ZAN Following the Acrosome Reaction in Mammalian Sperm

To clarify the presence of ZAN after induced acrosome reaction, co-labelling of ZAN and the acrosomal status was performed on sperm from rabbits, boars, and bulls. Following the acrosome reaction induction, ZAN was absent in a subset of spermatozoa in all three species analyzed (Figure 4). Specifically, the absence of ZAN was detected in 33.62% of rabbit spermatozoa, 16.30% of boar spermatozoa, and 53.14% of bull spermatozoa. Conversely, spermatozoa that retained ZAN after the acrosome reaction exhibited localization predominantly within the post-acrosomal domain, accounting for 66.38% in rabbit (pattern 3 + pattern 4), 83.70% in boar, and 46.86% in bull spermatozoa. It is worth mentioning that 38.7% of rabbit spermatozoa also showed labelled equatorial bulges (pattern 3).

2.6. Quantitative Analysis of ZAN Protein

DIA-NN filtered reports were used for quantifying ZAN, in which abundance values represented median peak areas. ZAN was observed in every experimental condition for each species (Figure 5). While no significant differences were detected across conditions in rabbit and boar samples (one-way ANOVA, p > 0.05), bovine sperm showed significant variations (Kruskal-Wallis, p < 0.05). Specifically, rabbit samples exhibited an area of 360,601.00 in ejaculated sperm, 285,123.92 in selected sperm via swim-up in capacitation medium and 206,755.46 in acrosome-reacted sperm. Turning to boar samples, proteomic analysis revealed an area of 121,939.47 in ejaculated sperm. Following swim-up selection in capacitation medium, this value increased to 197,038.60, whereas induction of acrosome reaction resulted in a decrease to 154,760.00. Despite no significant differences were observed in boar sperm between experimental conditions, the high sensitivity of the technique revealed a slight tendency (one-way ANOVA, p = 0.065). A similar trend was observed in bull sperm samples, where the area increased significantly from 253,020.00 in ejaculated sperm to 287,136.00 after selection (Bonferroni corrected; p < 0.01), and declined significantly to 236,566.00 following acrosome reaction induction (Bonferroni corrected; p < 0.01).

3. Discussion

In the process of capacitation, a set of proteins are translocated from the acrosomal membrane to the anterior region of the plasma membrane with the purpose of recognizing the zona pellucida. According to Tanphaichitr (2015) [14], ZAN is one of the most abundant proteins within these protein complexes. In light of various studies that confirm its involvement in the recognition of zona pellucida, the original hypothesis of its involvement after the acrosome reaction was largely unexplored. However, it should be noted that there are proteins, such as CRISP1, that participate in sperm-zona pellucida binding and migrate to the equatorial region during the acrosome reaction, where they are involved in fusing the sperm and the oocyte [15]. Consequently, our group decided to investigate whether ZAN is redistributed after the acrosome reaction. From among the Boreoeutheria mammals, two species of Laurasiatheria (boar and bull) and one Euarchontoglires (rabbit) were selected for this study.

Similarly to the literature, we found ZAN in the anterior region of intact spermatozoa in all species studied. However, the use of an antibody directed at the MAM region rather than the regions known as ‘active’ D0-D3 allowed us to detect ZAN location in the post-acrosomal region of both intact and reacted spermatozoa. When we analyze intact spermatozoa (the majority group subjected to E and S conditions) we discovered different patterns of ZAN localization based on the species studied. In rabbits and bulls, ZAN can be found in the acrosomal region (apical ridge + pre-equatorial subdomain), either with or without the post-acrosomal region (Patterns 1 and 2, respectively). Meanwhile, intact boar spermatozoa showed ZAN in the pre-equatorial and post-acrosomal domains, with or without intense labeling along the apical ridge. This specific labelling of the apical zone is consistent with Tanphaichitr (2015) [14] model of protein relocation in pigs that interacts with the ZP. Moreover, this protein dynamic has also been observed in boars by our group with the IZUMO1 protein [13], which is involved in both adhesion and fusion of gametes.

With regard to the reacted spermatozoa, we observed that despite variable ZAN loss according to the species, it is largely retained in the post-acrosomal region (Figure 4). It is important to note that ZAN is found in some rabbit sperm in a symmetrical thickening above the equatorial segment (equatorial bulges, Pattern 3) [16]. This morphological specialization of the equatorial segment could increase the contact surface, facilitating adhesion and fusion of the sperm and oocyte membranes. In addition, it may help maintain proper orientation of the sperm when it contacts the oolema, promoting efficient fusion.

Complementarily, an exploratory assay performed on porcine spermatozoa (Video 1) revealed an asymmetric localization of ZAN, restricted to one side of the post-acrosomal region. This polarized distribution is particularly noteworthy considering the characteristic tangential interaction between mammalian spermatozoa and the oocyte. Rather than contacting the oolemma perpendicularly, mammalian spermatozoa are thought to maintain a defined dorsoventral orientation and establish membrane contact at an oblique angle [17]. The asymmetric localization of ZAN is therefore consistent with the existence of a dorsoventrally polarized sperm head and suggests that the protein is associated with the specialized membrane domain that engages the oolemma during the final stages of fertilization.

Quantitative proteomic profiling via LC–MS/MS corroborated our immunolocalization data, revealing distinct species-specific trajectories for ZAN during fertilization competence acquisition (Figure 5). While bull sperm exhibited a statistically significant increase in ZAN abundance following the swim-up selection in capacitation medium, boar samples showed a no significant but slight elevation consistent with effective selection. In contrast, swim-up selection in capacitation medium in rabbit sperm was a reduction in population variance rather than an enhancement of protein levels. Conversely, acrosome reaction induction triggered a consistent decline in ZAN abundance across all species. This quantitative reduction aligns with our fluorescence data (Figure 4), suggesting that the observed loss reflects protein redistribution or shedding consequent to acrosomal exocytosis.

Taken together, the fluorescence analyses, three-dimensional reconstruction, and proteomic data support a coherent model for the localization of ZAN during fertilization (Figure 6). This model illustrates, for the first time, the conserved retention of ZAN after the acrosome reaction and provides a conceptual framework linking its spatial distribution to its proposed role during the final stages of fertilization across rabbit, boar and bull.

4. Materials and Methods

4.1. Experimental Design

Ejaculates from three different species (boar, rabbit, and bull) were processed under the following conditions (Figure 1): ejaculated (E), selected via swim-up in capacitation medium (S), and acrosome reaction induced (AR). In all conditions, double labelling was performed to determine ZAN distribution and to classify the acrosomal status using PNA-FITC (acrosome-intact vs. acrosome-reacted). In the pre- and post-selection conditions (E and S), only spermatozoa with intact acrosomes were evaluated, whilst in the induced acrosomal reaction condition, acrosome-reacted spermatozoa were evaluated. Additionally, unfixed aliquots from each of the three conditions of each species were subjected to quantitative proteomic analysis by LC-MS/MS.

Figure 7. Schematic description of the experimental design followed. Ejaculated (E); selected via swim-up in capacitation medium (S); acrosome reaction induced (AR); paraformaldehyde (PFA).

4.2. Sample Preparation

Boar semen (n = 7; Pietrain, Axiom Genetics line; purchased from SPERMATICA REPRODUCCION SL., Lorca, Spain) was collected using the gloved hand technique and packaged in bags concentrated at 30 mill/mL in Zoosperm ND10 (TECNOVET SL., Barcelona, Spain). Rabbit semen (n = 7; New Zealand) was collected using artificial vagina in the animal facility of the University of Murcia. Commercial cryopreserved bull semen (n = 6; Limousina; Aberekin S.A., Bizkaia, Spain) was thawed by immersion in a 37ºC water bath for 30 sec. Prior to sample processing, the status of the spermatozoa was corroborated by studying the concentration and motility using the AI Station computer-assisted sperm analysis (CASA) system (Sperm Analysis Technologies S.L., Spermtech, Valencia, Spain) viability with VitalStain^TM (NidaCon International AB, Mölndal, Sweden), and morphology with SpermBlue (Microptic, Barcelona, Spain). Semen from each species was centrifugated and resuspended in non-capacitation medium at 10 mill/mL. The inclusion criteria established for using the ejaculates were: total motility >70%, viable sperm > 90%, and morpho-anomalies ≤ 30%.

4.3. Sperm Selection via Swim-Up Capacitation Medium

The swim-up selection [18] consisted of transferring 100 mL of the sample to the bottom of an Eppendorf tube containing 1 mL of capacitation medium (see Table A1). The sample was then incubated at the appropriate temperature (boar 38.5ºC, rabbit 37ºC and bull 38ºC) with 5% (v/v) CO₂ and 45º inclination for 1 h. Finally, the supernatant fraction (900 mL) was collected and washed three times with sterile-filtered Dulbecco’s phosphate buffered saline without calcium, magnesium, and phenol red (PBS, Capricorn Scientific GmbH, Ebsdorfergrund, Germany) by centrifugation (200 g, 5 min).

4.4. Induction and Evaluation of Acrosomal Status

Acrosome reaction was induced by adding 10 mM of calcium ionophore A23187 (Sigma-Aldrich^®, Saint Louis, MO, USA) and 2 mM of calcium chloride (Panreac Química S.L.U, Barcelona, Spain) at the appropriate temperature (boar 38.5ºC, rabbit 37ºC and bull 38ºC) with 5% (v/v) CO₂ for 1 h following previous protocols of our group [19,20].

The samples were fixed on coverslips with methanol for 30 min. Then, the cells were washed three times with phosphate-buffered saline (PBS) and incubated with Peanut agglutinin lectin conjugated with fluorescein-5-isothiocyanate (PNA-FITC, Vector Laboratories Burlingame, CA) at a final concentration of 5 mg/mL for 30 min. After three washes, the samples were mounted using Fluoroshield^TM with 4′,6-diamidine-2′-phenylindole dihydrochloride (Sigma-Aldrich^®, Saint Louis, MO, USA).

4.5. Fixation

All sperm physiological conditions (E, S, and AR) were divided into two aliquots. One of them was fixed in 2% (w/v) paraformaldehyde (Electron MicroscopySciences, Hatfield, PA, USA) diluted in PBS during 45 min at 4ºC. Afterward, the fixative solution was replaced with PBS to reach a final concentration of 10 mill/mL and the samples were stored at 4ºC until their use. The other aliquot was stored unfixed at -80ºC for proteomic analysis.

4.6. ZAN Immunostaining

A total of 5 μl of each paraformaldehyde-fixed sample were placed on a coverslip. When the smear was dry, cells were washed twice in PBS for 5 min. Then, the smears were permeabilized with 0.2% (w/v) Triton X-100 for 10 min and blocked for 15 min in 0.2% (w/v) BSA-PBS. Afterward, smears were incubated with anti-ZAN antibody (1:100) produced in rabbit (Biorbyt Ltd., Cambridge, United Kingdom) overnight at 4ºC. Subsequently, smears were washed and incubated with a secondary anti-rabbit antibody conjugated with Cy³ (1:100, Jackson ImmunoResearch, Ely, United Kingdom) for 1 h at room temperature (RT) in darkness. For the co-staining of ZAN and acrosome status, samples were first incubated overnight with anti-ZAN followed by 30 min incubation with PNA-FITC. Finally, the smears were washed and mounted using Fluoroshield^TM with DAPI. Negative controls were performed by omitting the primary antibody.

A minimum of 200 cells were evaluated in each condition using a Confocal Laser Scanning Zeiss LSM 800 Microscope (Zeiss, Oberkochen, Germany) with an oil 100x objective and 405 nm, 488 nm, and 561 nm lasers. The ZAN staining pattern in the sperm head and acrosome were quantified as percentages (%).

4.7. LC MS/MS Analysis

The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia. This proteomics laboratory is a member of Proteored. In-solution samples of six randomly selected males of each specie (rabbit, boar, and bull) under all experimental conditions (ejaculated spermatozoa, spermatozoa selected by swim-up in capacitation medium, and acrosome reaction-induced spermatozoa.) were used. The samples from rabbit and bull were processed following the LC-MS/MS workflow previously established for boar samples [13]. All analytical parameters and quality control criteria were maintained consistent with the original protocol to ensure methodological reproducibility across species.

4.8. Statistical Analysis

Statistical analyses were conducted using IBM SPSS Statistics 28.0 (IBM, Armonk, NY). The Shapiro-Wilk test showed that ZAN location was normally distributed for each sperm physiological condition (E and S) in boar and bull, whereas it was not normally distributed in rabbit. Homogeneity of variances was evaluated using Levene’s test when parametric analyses were applicable. Variables following normal distribution were analyzed by a student’s t-test (two tailed). In cases where the assumption of homogeneity of variance was violated, the Welch correction was applied. To ensure the robustness of the parametric findings, all comparisons conducted using t-tests were additionally verified using the nonparametric Mann–Whitney U test, yielding the same overall results. Variables not following a normal distribution were analyzed by nonparametric Mann–Whitney U tests. Statistical significance was set at p < 0.05 for all analyses. In the analysis of the area data obtained with DIA-NN, the rabbit and the boar showed normal distributions, but the bull did not. One-way ANOVA was used for species with normal distributions, whereas Kruskal-Wallis with post-hoc Bonferroni correction was used for species with nonnormal distributions.

5. Conclusions

Collectively, this multi-species approach provides evidence that ZAN retention after the acrosome reaction is conserved across mammalian spermatozoa. Rather than being completely released during acrosomal exocytosis, ZAN remains associated with membrane domains involved in the final stages of fertilization. Its persistence within the equatorial segment or post-acrosomal region, together with the asymmetric localization observed in porcine spermatozoa, is consistent with the functional polarization of the sperm head and suggests that ZAN contributes to the directional interaction between the sperm plasma membrane and the oolemma during gamete binding and/or fusion.

Author Contributions

Conceptualization, M.J.G.-T. and P.S-E.; methodology, M.H.-F., P.S-E. and L.D.-F; formal analysis, M.H.-F., investigation, M.H.-F., P.S-E., L.D.-F., N.F-C. and L.R-G.; data curation, M.H.-F., P.S-E., L.D.-F., N.F-C., L.R-G. and M.J.G.-T.; writing—original draft preparation, M.H.-F.; writing—review and editing, M.H.-F, M.J.G.-T. and P.S-E.; visualization, M.H.-F; supervision, M.J.G.-T. and P.S-E.; project administration, M.J.G.-T.; funding acquisition, M.J.G.-T., M.H.-F., L. R.-G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the project PID2021-123091NB-C22 funded by MCIN/AEI/10.13039/501100011033 y por FEDER Una manera de hacer Europa; and University of Alicante (VIGROB-367). Miranda Hernández-Falcó predoctoral contract is part of the grant PRE2022-101681, funded by MCIN/AEI/10.13039/501100011033 and FSE+. The APC was funded by University of Alicante (VIGROB-367).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data supporting the findings of this study are contained within the article.

Acknowledgments

This work is part of the doctoral thesis of Miranda Hernández-Falcó, developed within the PhD Program in Health Sciences at the University of Alicante.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:

AR	Acrosome reaction-induced spermatozoa
BSA	Bovine serum albumine
E	Ejaculated spermatozoa
PBS	Phosphate-buffered saline
PFA	Paraformaldehyde
S	Spermatozoa selected by swim-up in capacitation medium
ZAN	Zonadhesin
ZP	Zona pellucida

Appendix A

Table A1. TALP medium composition.

Reagent	Rabbit	Boar	Bull
NaCl	114.06 mM	114.06 mM	114.06 mM
KCl	3.20 mM	3.20 mM	3.20 mM
NaH₂PO₄	0.35 mM	0.35 mM	0.35 mM
CaCl₂	-	-	2.10 mM
MgCl₂6H₂O	0.50 mM	0.5 mM	0.50 mM
Glucose	5.00 mM	5.00 mM	-
NaHCO₃	25.07 mM	25.07 mM	-
Caffeine	2 mM	2.00 mM	-
C₆H₁₀CaO₆·5H₂O	8.00 mM	8.00 mM	-
Na pyruvate	1.10 mM	1.10 mM	1.10 mM
CH₃CH(OH)COONa	18.00 mM	18 mM	18.00 mM
NaC₃H₅O	-	10 mM	-
PVA	1.00 mg mL⁻¹	1.00 mg mL⁻¹	-
Kanamycin sulphate	0.17 mM	0.17 mM	0.17 mM
Red phenol	0.003 mM	0.003 mM	0.003 mM
BSA	5.00 mg mL⁻¹	3.00 mg mL⁻¹	3.00 mg mL⁻¹
Heparin			0.01 mg mL⁻¹

TALP medium was previously pre-equilibrated overnight at the appropriate temperature, 5% (v/v) CO2, 95% humidified air and with a final pH of 7.4. All those reagents were purchased to Sigma (Sigma-Aldrich^®, Saint Louis, MO, USA) and were embryo and/or cell culture tested.

References

Hardy, D.M.; Garbers, D.L. A Sperm Membrane Protein That Binds in a Species-Specific Manner to the Egg Extracellular Matrix Is Homologous to von Willebrand Factor. J. Biol. Chem. 1995, 270, 26025–26028. [Google Scholar] [CrossRef]
Herlyn, H.; Zischler, H. The Molecular Evolution of Sperm Zonadhesin. Int. J. Dev. Biol. 2008, 52, 781–790. [Google Scholar] [CrossRef] [PubMed]
Hickox, J.R.; Bi, M.; Hardy, D.M. Heterogeneous Processing and Zona Pellucida Binding Activity of Pig Zonadhesin. J. Biol. Chem. 2001, 276, 41502–41509. [Google Scholar] [CrossRef] [PubMed]
Bi, M.; Hickox, J.R.; Winfrey, V.P.; Olson, G.E.; Hardy, D.M. Processing, Localization and Binding Activity of Zonadhesin Suggest a Function in Sperm Adhesion to the Zona Pellucida during Exocytosis of the Acrosome. Biochem. J. 2003, 375, 477–488. [Google Scholar] [CrossRef] [PubMed]
Gao, Z.; Garbers, D.L. Species Diversity in the Structure of Zonadhesin, a Sperm-Specific Membrane Protein Containing Multiple Cell Adhesion Molecule-like Domains. J. Biol. Chem. 1998, 273, 3415–3421. [Google Scholar] [CrossRef] [PubMed]
Tardif, S.; Wilson, M.D.; Wagner, R.; Hunt, P.; Gertsenstein, M.; Nagy, A.; Lobe, C.; Koop, B.F.; Hardy, D.M. Zonadhesin Is Essential for Species Specificity of Sperm Adhesion to the Egg Zona Pellucida. J. Biol. Chem. 2010, 285, 24863–24870. [Google Scholar] [CrossRef] [PubMed]
Gao, Z.; Harumi, T.; Garbers, D.L. Chromosome Localization of the Mouse Zonadhesin Gene and the Human Zonadhesin Gene (ZAN). Genomics 1997, 41, 119–122. [Google Scholar] [CrossRef] [PubMed]
Lea, I.A.; Sivashanmugam, P.; O’Rand, M.G. Zonadhesin: Characterization, Localization, and Zona Pellucida Binding1. Biol. Reprod. 2001, 65, 1691–1700. [Google Scholar] [CrossRef] [PubMed]
Olson, G.E.; Winfrey, V.P.; Bi, M.; Hardy, D.M.; NagDas, S.K. Zonadhesin Assembly into the Hamster Sperm Acrosomal Matrix Occurs by Distinct Targeting Strategies during Spermiogenesis and Maturation in the Epididymis. Biol. Reprod. 2004, 71, 1128–1134. [Google Scholar] [CrossRef] [PubMed]
Tardif, S.; Cormier, N. Role of Zonadhesin during Sperm-Egg Interaction: A Species-Specific Acrosomal Molecule with Multiple Functions. Mol. Hum. Reprod. 2011, 17, 661–668. [Google Scholar] [CrossRef] [PubMed]
Teves, M.E.; Roldan, E.R.S. Spermbauplan and Function and Underlying Processes of Sperm Formation and Selection. Physiol. Rev. 2022, 102, 7–60. [Google Scholar] [CrossRef] [PubMed]
Stival, C.; Puga Molina, L. del C.; Paudel, B.; Buffone, M.G.; Visconti, P.E.; Krapf, D. Sperm Capacitation and Acrosome Reaction in Mammalian Sperm. In Sperm Acrosome Biogenesis and Function During Fertilization; Buffone, M.G., Ed.; Springer, 2016; Vol. 220, pp. 93–106. [Google Scholar]
Hernández-Falcó, M.; Sáez-Espinosa, P.; López-Botella, A.; Robles-Gómez, L.; García-Vázquez, F.A.; Izquierdo-Rico, M.J.; Llamas-López, P.J.; Gómez-Torres, M.J. Immunolocalization and Proteomic Analyses of IZUMO1 in Porcine Spermatozoa. Front. Cell Dev. Biol. 2025, 13. [Google Scholar] [CrossRef] [PubMed]
Tanphaichitr, N.; Kongmanas, K.; Kruevaisayawan, H.; Saewu, A.; Sugeng, C.; Fernandes, J.; Souda, P.; Angel, J.B.; Faull, K.F.; Aitken, R.J.; et al. Remodeling of the Plasma Membrane in Preparation for Sperm-Egg Recognition: Roles of Acrosomal Proteins. In Proceedings of the Asian Journal of Andrology; Medknow Publications, 1 July 2015; Vol. 17, pp. 574–582. [Google Scholar]
Cohen, D.J.; Maldera, J.A.; Vasen, G.; Ernesto, J.I.; Muñoz, M.W.; Battistone, M.A.; Cuasnicú, P.S. Epididymal Protein CRISP1 Plays Different Roles during the Fertilization Process. J. Androl. 2011, 32, 672–678. [Google Scholar] [CrossRef] [PubMed]
Bernstein, M.H.; Teichman, R.J. Regional Differentiation in the Heads of Spermatozoa of Rabbit, Man and Bull. Am. J. Anat. 1972, 133, 165–178. [Google Scholar] [CrossRef] [PubMed]
Bedford, J.M. Why Do Penetrating Sperm Create an Oblique Path in the Zona Pellucida. Reproduction 2006, 131, 23–25. [Google Scholar] [CrossRef] [PubMed]
Boiti, C.; Castellini, C.; Theau-Clement, M.; Besenfelder, U.; Liguori, L.; Renieri, T.; Pizzi, F. Guidelines for the Handing of Rabbit Bucks and Semen. World Sci. Reprod. 2005, 13, 71–91. [Google Scholar]
Robles-Gómez, L.; González-Brusi, L.; Sáez-Espinosa, P.; Huerta-Retamal, N.; Cots-Rodríguez, P.; Avilés, M.; Gómez-Torres, M.J. Specific Lectin Binding Sites during in Vitro Capacitation and Acrosome Reaction in Boar Spermatozoa. Ital. J. Anim. Sci. 2021, 20, 372–382. [Google Scholar] [CrossRef]
Sáez-Espinosa, P.; Torrijo-Boix, S.; Huerta-Retamal, N.; Avilés, M.; Aizpurua, J.; Romero, A.; Gómez-Torres, M.J. La Capacitación y La Reacción Acrosómica Se Asocian Con Cambios En La Localización Del Ácido Siálico y En La Morfometría de La Región Cefálica Del Espermatozoide Humano. Rev. Int. Androl. 2018, 16, 20–27. [Google Scholar] [CrossRef] [PubMed]

Figure 1. Analysis and distribution of ZAN staining patterns in rabbit sperm. (a) Representative images of ZAN and acrosome status co-staining. (b) Percentages of ZAN staining patterns in ejaculated sperm (E) and selected via swim-up in capacitation medium (S). P1, speckled staining in the apical ridge and pre-equatorial subdomain; P2, strong apical ridge and pre-equatorial subdomain staining with speckled staining in the post-acrosomal domain; P3 staining at the equatorial bulges as well as post-acrosomal domain; P4, staining only at the post-acrosomal domain; P5, without labelling. Scale bar: 10 μm, common to all images. Significant differences at p < 0.001 (***) level according to Mann–Whitney U test.

Figure 2. Analysis and distribution of ZAN staining patterns in boar sperm. (a) Representative images of ZAN and acrosome status co-staining. (b) Percentages of ZAN staining patterns in ejaculated sperm (E) and selected via swim-up in capacitation medium (S). P1, speckled staining in the pre-equatorial subdomain and post-acrosomal domain; P2, strong apical ridge staining with speckled staining in the pre-equatorial subdomain and post-acrosomal domain; P3, speckled staining in the post-acrosomal domain; P4, without labelling. Scale bar: 5 μm, common to all images. Statistical significance was assessed using Student’s t-tests or Welch’s t-test, as determined by homogeneity of variances (Levene’s test). Significant differences at p < 0.05 (*) and p < 0.001 (***).

Figure 3. Analysis and distribution of ZAN staining patterns in bull sperm. (a) Representative images of ZAN and acrosome status co-staining. (b) Percentages of ZAN staining patterns in ejaculated sperm (E) and selected via swim-up in capacitation medium (S). P1, speckled staining in the apical ridge and pre-equatorial subdomain; P2, strong apical ridge and pre-equatorial subdomain staining with speckled staining in the post-acrosomal domain; P3, staining only at the post-acrosomal domain; P4, without labelling. Statistical significance was assessed using Student’s t-tests or Welch’s t-test, as determined by homogeneity of variances (Levene’s test). Significant differences at p < 0.01 (**).

Figure 4. ZAN distribution following the acrosome reaction in rabbit, boar, and bull spermatozoa.

Figure 5. Relative abundance of ZAN across ejaculated sperm (E), selected via swim-up in capacitation medium (S) and acrosome reaction induced (AR) states in rabbit, boar, and bull sperm. Data represent median peak area. Statistical significance was assessed using one-way ANOVA (rabbit, boar) or Kruskal-Wallis test with Bonferroni correction (bovine), as determined by normality assumptions (Shapiro-Wilk test). Significant differences at p < 0.01 (**) in bovine samples between ejaculated vs. selected; and selected vs. acrosome-reacted.

Video 1. Three-dimensional fluorescence reconstruction showing ZAN localization in porcine spermatozoa. Generated from fluorescence microscopy Z-stack images showing the sperm nucleus (blue) and ZAN localization (red). ZAN is detected throughout the acrosomal region and is also present on one side of the post-acrosomal region.

Figure 6. Proposed model of ZAN localization during mammalian fertilization in rabbit, boar, and bull. (1) Before capacitation, ZAN is localized in the pre-equatorial region, specifically in the outer acrosomal membrane and, depending on the species, may also be present in the post-acrosomal region. (2) Following sperm capacitation, ZAN becomes exposed at the sperm surface, allowing its interaction with the zona pellucida. (3) After acrosomal exocytosis, ZAN remains on the ventral side of the post-acrosomal region, where it may participate in the interaction between the sperm plasma membrane and the oolemma.

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Copyright: This open access article is published under a Creative Commons CC BY 4.0 license, which permit the free download, distribution, and reuse, provided that the author and preprint are cited in any reuse.