Positive selection in gamete interaction proteins in Carnivora.

The absence of robust interspecific isolation barriers among pantherines, including the iconic South American jaguar (Panthera onca), led us to study molecular evolution of typically rapidly evolving reproductive proteins within this subfamily and related groups. In this study, we delved into the evolutionary forces acting on the zona pellucida (ZP) gamete interaction protein family and the sperm-oocyte fusion protein pair IZUMO1-JUNO across the Carnivora order, distinguishing between Caniformia and Feliformia suborders and anticipating few significant diversifying changes in the Pantherinae subfamily. A chromosome-resolved jaguar genome assembly facilitated coding sequences, enabling the reconstruction of protein evolutionary histories. Examining sequence variability across more than 30 Carnivora species revealed that Feliformia exhibited significantly lower diversity compared to its sister taxa, Caniformia. Molecular evolution analyses of ZP2 and ZP3, subunits directly involved in sperm-recognition, unveiled diversifying positive selection in Feliformia, Caniformia and Pantherinae, although no significant changes were linked to sperm binding. Structural cross-linking ZP subunits, ZP4 and ZP1 exhibited lower levels or complete absence of positive selection. Notably, the fusion protein IZUMO1 displayed prominent positive selection signatures and sites in basal lineages of both Caniformia and Feliformia, extending along the Caniformia subtree but absent in Pantherinae. Conversely, JUNO did not exhibit any positive selection signatures across tested lineages and clades. Eight Caniformia-specific positive selected sites in IZUMO1 were detected within two JUNO-interaction clusters. Our findings provide for the first time insights into the evolutionary trajectories of ZP proteins and the IZUMO1-JUNO gamete interaction pair within the Carnivora order.

Spermatozoa lacking Fertilization Influencing Membrane Protein (FIMP) fail to fuse with oocytes in mice.

Sperm-oocyte fusion is a critical event in mammalian fertilization, categorized by three indispensable proteins. Sperm membrane protein IZUMO1 and its counterpart oocyte membrane protein JUNO make a protein complex allowing sperm to interact with the oocyte, and subsequent sperm-oocyte fusion. Oocyte tetraspanin protein CD9 also contributes to sperm-oocyte fusion. However, the fusion process cannot be explained solely by these three essential factors. In this study, we focused on analyzing a testis-specific gene 4930451I11Rik and generated mutant mice using the CRISPR/Cas9 system. Although IZUMO1 remained in 4930451I11Rik knockout (KO) spermatozoa, the KO spermatozoa were unable to fuse with oocytes and the KO males were severely subfertile. 4930451I11Rik encodes two isoforms: a transmembrane (TM) form and a secreted form. Both CRISPR/Cas9-mediated TM deletion and transgenic (Tg) rescue with the TM form revealed that only the TM form plays a critical role in sperm-oocyte fusion. Thus, we renamed this TM form Fertilization Influencing Membrane Protein (FIMP). The mCherry-tagged FIMP TM form was localized to the sperm equatorial segment where the sperm-oocyte fusion event occurs. Thus, FIMP is a sperm-specific transmembrane protein that is necessary for the sperm-oocyte fusion process.

Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm-oocyte fusion in mice.

Sperm-oocyte membrane fusion is one of the most important events for fertilization. So far, IZUMO1 and Fertilization Influencing Membrane Protein (FIMP) on the sperm membrane and CD9 and JUNO (IZUMO1R/FOLR4) on the oocyte membrane have been identified as fusion-required proteins. However, the molecular mechanisms for sperm-oocyte fusion are still unclear. Here, we show that testis-enriched genes, sperm-oocyte fusion required 1 (Sof1/Llcfc1/1700034O15Rik), transmembrane protein 95 (Tmem95), and sperm acrosome associated 6 (Spaca6), encode sperm proteins required for sperm-oocyte fusion in mice. These knockout (KO) spermatozoa carry IZUMO1 but cannot fuse with the oocyte plasma membrane, leading to male sterility. Transgenic mice which expressed mouse Sof1, Tmem95, and Spaca6 rescued the sterility of Sof1, Tmem95, and Spaca6 KO males, respectively. SOF1 and SPACA6 remain in acrosome-reacted spermatozoa, and SPACA6 translocates to the equatorial segment of these spermatozoa. The coexpression of SOF1, TMEM95, and SPACA6 in IZUMO1-expressing cultured cells did not enhance their ability to adhere to the oocyte membrane or allow them to fuse with oocytes. SOF1, TMEM95, and SPACA6 may function cooperatively with IZUMO1 and/or unknown fusogens in sperm-oocyte fusion.

The Role of Sperm Proteins IZUMO1 and TMEM95 in Mammalian Fertilization: A Systematic Review.

Gamete membrane fusion is a critical cellular event in sexual reproduction. In addition, the generation of knockout models has provided a powerful tool for testing the functional relevance of proteins thought to be involved in mammalian fertilization, suggesting IZUMO1 and TMEM95 (transmembrane protein 95) as essential proteins. However, the molecular mechanisms underlying the process remain largely unknown. Therefore, the aim of this study was to summarize the current knowledge about IZUMO1 and TMEM95 during mammalian fertilization. Hence, three distinct databases were consulted-PubMed, Scopus and Web of Science-using single keywords. As a result, a total of 429 articles were identified. Based on both inclusion and exclusion criteria, the final number of articles included in this study was 103. The results showed that IZUMO1 is mostly studied in rodents whereas TMEM95 is studied primarily in bovines. Despite the research, the topological localization of IZUMO1 remains controversial. IZUMO1 may be involved in organizing or stabilizing a multiprotein complex essential for the membrane fusion in which TMEM95 could act as a fusogen due to its possible interaction with IZUMO1. Overall, the expression of these two proteins is not sufficient for sperm-oocyte fusion; therefore, other molecules must be involved in the membrane fusion process.

Sperm-oocyte signaling: the role of IZUMO1R and CD9 in PTK2B activation and actin remodeling at the sperm binding site†.

Sperm-oocyte binding initiates an outside-in signaling event in the mouse oocyte that triggers recruitment and activation of the cytosolic protein kinase PTK2B in the cortex underlying the bound sperm. While not involved in gamete fusion, PTK2B activity promotes actin remodeling events important during sperm incorporation. However, the mechanism by which sperm-oocyte binding activates PTK2B is unknown, and the present study examined the possibility that sperm interaction with specific oocyte surface proteins plays an important role in PTK2B activation. Imaging studies revealed that as IZUMO1R and CD9 became concentrated at the sperm binding site, activated (phosphorylated) PTK2B accumulated in the cortex underlying the sperm head and in microvilli partially encircling the sperm head. In order to determine whether IZUMO1R and/or CD9 played a significant role in PTK2B recruitment and activation at the sperm binding site, the ability of oocytes null for Izumo1r or Cd9, to initiate an increase in PTK2B content and activation was tested. The results revealed that IZUMO1R played a minor role in PTK2B activation and had no effect on actin remodeling; however, CD9 played a very significant role in PTK2B activation and subsequent actin remodeling at the sperm binding site. These findings suggest the possibility that interaction of sperm surface proteins with CD9 or CD9-associated oocyte proteins triggers PTK2B activation at the sperm binding site.

Cleavage of SPACA1 regulates assembly of sperm-egg membrane fusion machinery in mature spermatozoa†.

The acrosome reaction is a multi-step event essential for physiological fertilization. During the acrosome reaction, gamete fusion-related factor IZUMO1 translocates from the anterior acrosome to the equatorial segment and assembles the gamete fusion machinery. The morphological changes in the acrosome reaction process have been well studied, but little is known about the molecular mechanisms of acrosome reorganization essential for physiological gamete membrane fusion. To elucidate the molecular mechanisms of IZUMO1 translocation, the steps of the acrosome reaction during that process must be clarified. In this study, we established a method to detect the early steps of the acrosome reaction and subdivided the process into seven populations through the use of two epitope-defined antibodies, anti-IZUMO1 and anti-SPACA1, a fertilization-inhibiting antibody. We found that part of the SPACA1 C-terminus in the periacrosomal space was cleaved and had begun to disappear when the vesiculation of the anterior acrosome occurred. The IZUMO1 epitope externalized from the acrosomal lumen before acrosomal vesiculation and phosphorylation of IZUMO1 occurred during the translocation to the equatorial segment. IZUMO1 circumvented the area of the equatorial segment where the SPACA1C-terminus was still localized. We therefore propose an IZUMO1 translocation model and involvement of SPACA1.

Sperm IZUMO1-Dependent Gamete Fusion Influences Male Fertility in Mice.

Sperm-egg fusion is accomplished through the interaction of a specific set of membrane proteins in each gamete: sperm IZUMO1 and oocyte JUNO. Recently, we found that alternative splicing of the Izumo1 gene generates a novel IZUMO1 isoform (IZUMO1_v2). Here, we obtained four mouse lines, having graded different levels of IZUMO1 protein by combining an original IZUMO1 (IZUMO1_v1) knockout with IZUMO1-null (both IZUMO1_v1 and _v2 disrupted) genetic background, in order to determine how the quantity of IZUMO1 influences male fertility. Subsequently, we clarified that the signal intensity from two quantitative assays, western blot and immunostaining analyses with a monoclonal antibody against mouse IZUMO1, were strongly correlated with average litter size. These results suggest that evaluating IZUMO1 protein levels is useful for predicting fecundity, and is a suitable test for male fertility.

Mutational analysis of IZUMO1R in women with fertilization failure and polyspermy after in vitro fertilization.

PURPOSE: The etiology of fertilization failure and polyspermy during assisted reproductive technology (ART) remains elusive. The aim of this study was to determine whether mutations in the IZUMO1 receptor (IZUMO1R) gene, which is essential for mammalian fertilization, contribute to the pathogenesis of fertilization failure or polyspermy in humans. METHODS: We recruited 215 female subjects with fertilization failure/poor fertilization, 330 females with polyspermy, and 300 matched controls. All subjects underwent IVF treatment. Peripheral blood DNA of cases was extracted and screened for mutations in IZUMO1R gene. RESULTS: Four rare single nucleotide polymorphisms (SNPs) of the IZUMO1R were identified among specimens from patients with fertilization failure and polyspermy but were absent in the 300 control subjects. These included a missense SNP (rs76779571 in exon 4), which was found in two fertilization failure patients, and a nonsynonymous SNP (rs61742524 in exon 1) and two synonymous SNPs (rs76781645 in exon 1 and rs377369966 in intron 2), which were found among three polyspermy cases. CONCLUSIONS: The variations in IZUMO1R might play a role in the pathogenesis of fertilization failure and polyspermy, and the putative functions and effects of these rare variants require further studies.

Binding of sperm protein Izumo1 and its egg receptor Juno drives Cd9 accumulation in the intercellular contact area prior to fusion during mammalian fertilization.

Little is known about the molecular mechanisms that induce gamete fusion during mammalian fertilization. After initial contact, adhesion between gametes only leads to fusion in the presence of three membrane proteins that are necessary, but insufficient, for fusion: Izumo1 on sperm, its receptor Juno on egg and Cd9 on egg. What happens during this adhesion phase is a crucial issue. Here, we demonstrate that the intercellular adhesion that Izumo1 creates with Juno is conserved in mouse and human eggs. We show that, along with Izumo1, egg Cd9 concomitantly accumulates in the adhesion area. Without egg Cd9, the recruitment kinetics of Izumo1 are accelerated. Our results suggest that this process is conserved across species, as the adhesion partners, Izumo1 and its receptor, are interchangeable between mouse and human. Our findings suggest that Cd9 is a partner of Juno, and these discoveries allow us to propose a new model of the molecular mechanisms leading to gamete fusion, in which the adhesion-induced membrane organization assembles all key players of the fusion machinery.

Effects of latrunculin A on the relocation of sperm IZUMO1 during gamete interaction in mouse.

The sperm protein IZUMO1 plays a central role in gamete fusion. In mouse sperm, IZUMO1 is enriched at the acrosomal cap before the acrosome reaction, and at the equatorial segment following this reaction; its relocation is dependent on filamentous actin. How actin polymerization affects IZUMO1 relocation during gamete interaction remains unknown. The present study addressed these processes using latrunculin A (LatA), an inhibitor of actin polymerization. We report that 25 µM LatA blocked actin polymerization in the capacitated sperm head, resulting in a marked decrease in sperm with relocated IZUMO1 during the A23187-induced acrosome reaction and cumulus layer penetration. Treated sperm also exhibited reduced zona pellucida penetration and fertilizing capacity. Interestingly, LatA-treated sperm present in the perivitelline space of eggs did not show impaired IZUMO1 relocation. Thus, IZUMO1 relocation represents one method by which eggs may select for or rescue sperm that are competent to undergo gamete adhesion/fusion. These data support the hypothesis that dynamic movement of IZUMO1 is essential for gamete fusion during mouse fertilization.

Novel insights into the molecular mechanism of sperm-egg fusion via IZUMO1.

When a spermatozoon fertilizes an oocyte in mammals, there must be an extremely precise regulation system for successful gamete fusion to occur, which is the final step of fertilization. Using gene-modified animals, IZUMO1 on the sperm side and its receptor, JUNO, on the ovum side, have been unveiled as indispensable factors for triggering membrane fusion. We recently analyzed the detailed molecular machinery of the IZUMO1-JUNO recognition system and clarified the tertiary architecture of the IZUMO1-JUNO complex based on the crystal structure. Over the past 2 years, important discoveries have successively emerged, presenting a new perspective on fertilization. In this mini-review, I will initially explain the historical background of the molecular mechanism study of gamete fusion, and go on to describe our latest study data.

GPI-AP release in cellular, developmental, and reproductive biology.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) contain a covalently linked GPI anchor located on outer cell membranes. GPI-APs are ubiquitously conserved from protozoa to vertebrates and are critical for physiological events such as development, immunity, and neurogenesis in vertebrates. Both membrane-anchored and soluble GPI-APs play a role in regulating their protein conformation and functional properties. Several pathways mediate the release of GPI-APs from the plasma membrane by vesiculation or cleavage. Phospholipases and putative substrate-specific GPI-AP-releasing enzymes, such as NOTUM, glycerophosphodiesterase 2, and angiotensin-converting enzyme, have been characterized in mammals. Here, the protein modifications resulting from the cleavage of the GPI anchor are discussed in the context of its physiological functions.

The cell biology of mammalian fertilization.

Fertilization is the process by which eggs and spermatozoa interact, achieve mutual recognition, and fuse to create a zygote, which then develops to form a new individual, thus allowing for the continuity of a species. Despite numerous studies on mammalian fertilization, the molecular mechanisms underpinning the fertilization event remain largely unknown. However, as I summarize here, recent work using both gene-manipulated animals and in vitro studies has begun to elucidate essential sperm and egg molecules and to establish predictive models of successful fertilization.

Molecular dissection of IZUMO1, a sperm protein essential for sperm-egg fusion.

Although the membrane fusion of spermatozoon and egg cells is the central event of fertilization, the underlying molecular mechanism remains virtually unknown. Gene disruption studies have showed that IZUMO1 on spermatozoon and CD9 on oocyte are essential transmembrane proteins in sperm-egg fusion. In this study, we dissected IZUMO1 protein to determine the domains that were required for the function of sperm-egg fusion. We found that a fragment of the N terminus (Asp5 to Leu113) interacts with fertilization inhibitory antibodies. It also binds to the egg surface and effectively inhibits fusion in vitro. We named this fragment 'IZUMO1 putative functional fragment (IZUMO1PFF)'. Surprisingly, IZUMO1PPF still maintains binding ability on the egg surface of Cd9(-/-) eggs. A series of biophysical measurements using circular dichroism, sedimentation equilibrium and small angle X-ray scattering revealed that IZUMO1PFF is composed of an N-terminal unfolded structure and a C-terminal ellipsoidal helix dimer. Egg binding and fusion inhibition were not observed in the IZUMO1PFF derivative, which was incapable of helix formation. These findings suggest that the formation of a helical dimer at the N-terminal region of IZUMO1 is required for its function. Cos-7 cells expressing the whole IZUMO1 molecule bound to eggs, and IZUMO1 accumulated at the interface between the two cells, but fusion was not observed. These observations suggest that IZUMO1 alone cannot promote sperm-egg membrane fusion, but it works as a factor that is related to the cellular surface interaction, such as the tethering of the membranes by a helical region corresponding to IZUMO1PFF-core.

The challenges involved in elucidating the molecular basis of sperm-egg recognition in mammals and approaches to overcome them.

Sexual reproduction is used by many different organisms to create a new generation of genetically distinct progeny. Cells originating from separate sexes or mating types segregate their genetic material into haploid gametes which must then recognize and fuse with each other in a process known as fertilization to form a diploid zygote. Despite the central importance of fertilization, we know remarkably little about the molecular mechanisms that are involved in how gametes recognize each other, particularly in mammals, although the proteins that are displayed on their surfaces are almost certainly involved. This paucity of knowledge is largely due to both the unique biological properties of mammalian gametes (sperm and egg) which make them experimentally difficult to manipulate, and the technical challenges of identifying interactions between membrane-embedded cell surface receptor proteins. In this review, we will discuss our current knowledge of animal gamete recognition, highlighting where important contributions to our understanding were made, why particular model systems were helpful, and why progress in mammals has been particularly challenging. We discuss how the development of mammalian in vitro fertilization and targeted gene disruption in mice were important technological advances that triggered progress. We argue that approaches employed to discover novel interactions between cell surface gamete recognition proteins should account for the unusual biochemical properties of membrane proteins and the typically highly transient nature of their interactions. Finally, we describe how these principles were applied to identify Juno as the egg receptor for sperm Izumo1, an interaction that is essential for mammalian fertilization.

Identification of bovine sperm acrosomal proteins that interact with a 32-kDa acrosomal matrix protein.

Mammalian fertilization is accomplished by the interaction between sperm and egg. Previous studies from this laboratory have identified a stable acrosomal matrix assembly from the bovine sperm acrosome termed the outer acrosomal membrane-matrix complex (OMC). This stable matrix assembly exhibits precise binding activity for acrosin and N-acetylglucosaminidase. A highly purified OMC fraction comprises three major (54, 50, and 45 kDa) and several minor (38-19 kDa) polypeptides. The set of minor polypeptides (38-19 kDa) termed "OMCrpf polypeptides" is selectively solubilized by high-pH extraction (pH 10.5), while the three major polypeptides (55, 50, and 45 kDa) remain insoluble. Proteomic identification of the OMC32 polypeptide (32 kDa polypeptide isolated from high-pH soluble fraction of OMC) yielded two peptides that matched the NCBI database sequence of acrosin-binding protein. Anti-OMC32 recognized an antigenically related family of polypeptides (OMCrpf polypeptides) in the 38-19-kDa range with isoelectric points ranging between 4.0 and 5.1. Other than glycohydrolases, OMC32 may also be complexed to other acrosomal proteins. The present study was undertaken to identify and localize the OMC32 binding polypeptides and to elucidate the potential role of the acrosomal protein complex in sperm function. OMC32 affinity chromatography of a detergent-soluble fraction of bovine cauda sperm acrosome followed by mass spectrometry-based identification of bound proteins identified acrosin, lactadherin, SPACA3, and IZUMO1. Co-immunoprecipitation analysis also demonstrated the interaction of OMC32 with acrosin, lactadherin, SPACA3, and IZUMO1. Our immunofluorescence studies revealed the presence of SPACA3 and lactadherin over the apical segment, whereas IZUMO1 is localized over the equatorial segment of Triton X-100 permeabilized cauda sperm. Immunoblot analysis showed that a significant portion of SPACA3 was released after the lysophosphatidylcholine (LPC)-induced acrosome reaction, whereas the IZUMO1 and lactadherin polypeptides remain associated to the particulate fraction. Almost entire population of bovine sperm IZUMO1 relocates to the equatorial segment during the LPC-induced acrosome reaction. We propose that the interaction of OMC32 matrix polypeptide with detergent-soluble acrosomal proteins regulates the release of hydrolases/other acrosomal protein(s) during the acrosome reaction.

Does combining magnetic-activated cell sorting with density gradient or swim-up improve sperm selection?

PURPOSE: The present study aimed to evaluate whether combining the magnetic-activated cell sorting (MACS) with density-gradient (DG) or swim-up (SU) sperm separation techniques can improve sperm selection to obtain higher quality spermatozoa. METHODS: Two commonly used sperm selection techniques, SU and DG, were compared to MACS combined with either SU or DG. Spermatozoa obtained from normozoospermic (n = 10) and oligozoospermic (n = 10) cases were grouped as SU, DG, SU+MACS, and DG+MACS followed by the analysis of sperm morphology, motility, DNA integrity, and the levels of Izumo-1 and PLCZ proteins. RESULTS: Although spermatozoa obtained by SU or DG when combined with MACS have improved aspects when compared to SU or DG alone, results did not reach a statistically significant level. Moreover, separation with MACS caused a significant loss in the numbers of total and rapid progressive spermatozoa. CONCLUSIONS: Considering the cost/benefit ratio, MACS application together with traditional techniques may only be preferred in certain cases having higher concentrations of spermatozoa, but it does not seem to be an ideal and practical sperm selection technique for routine use.

Coevolution of positively selected IZUMO1 and CD9 in rodents: evidence of interaction between gamete fusion proteins?

Proteins involved in sexual reproduction are known to evolve rapidly, often as the result of positive Darwinian selection, although the selective forces driving such adaptive changes are poorly understood. A process of coevolution between proteins in male and female gametes may promote rapid divergence of fertilization proteins. In the mouse, only two proteins have been shown so far to be essential for sperm-egg fusion, IZUMO1 in the sperm cell and CD9 in the egg. The role of these proteins has not been fully elucidated, and it has been suggested that they may act as fusogens, interacting in trans with proteins on the other cell, or regulators of fusogens through cis interactions. Here we analyze the evolution of IZUMO1 and CD9 in a group of rodent species. To assess possible protein interactions between IZUMO1 and CD9, we examined potential coevolution based on analyses of correlated evolutionary rates. We found evidence that both proteins evolve adaptively, with a more intense signal of positive selection in IZUMO1. In addition, our findings suggest that these proteins may have some form of interaction, although they have not been regarded as fusogens interacting directly with each other. The adaptive divergence of IZUMO1 and CD9 could influence reproductive compatibility, and, thus, these proteins may participate in the establishment of specific sperm-egg recognition systems. Further studies are required to uncover the role of IZUMO1 and CD9 during gamete fusion in order to understand the molecular basis of their coevolution, as other selective forces could also lead to general signatures of coevolution.

Expression of IZUMO1 and JUNO in the gonads of domestic cats (Felis catus).

Because of the time-consuming nature of surgical neutering and the rapid rate of reproduction among domestic cats, it is crucial to investigate alternative, nonsurgical methods of contraception for this species. Sperm protein IZUMO1 and its oocyte receptor JUNO have been proposed as potential targets for nonsurgical contraceptives. This study aimed to demonstrate (1) the protein coding sequence of feline IZUMO1 and JUNO, (2) gene expression in specific organs by measuring mRNA levels in different visceral tissues, and (3) the expression of IZUMO1 and JUNO during sperm maturation and folliculogenesis, respectively. Amplification for sequencing of feline IZUMO1 and JUNO was performed using the RT-PCR method. Levels of gene expression in different tissues were evaluated using real-time PCR. In situ hybridization was performed to localize JUNO mRNA in ovarian tissues. The complete coding sequences of IZUMO1 and JUNO were obtained and analyzed. A comparison between protein orthologs demonstrated the conservation of IZUMO1 and JUNO in Felidae. The real-time PCR results from various visceral organs indicated that IZUMO1 was significantly higher in the testis than in other organs, whereas JUNO was significantly higher in the ovary than in other organs. Expression of IZUMO1 was found to be higher in the testes than in the caput, corpus, and cauda of epididymides. In situ hybridization revealed that JUNO mRNA was in the ooplasm and nucleus of the primordial, primary, secondary, and antral follicles. Importantly, this was the first study to demonstrate the IZUMO1 and JUNO genes in the testis and ovary of cats. The results are useful for future research related to these genes and for developing contraceptives against these targets.

Detection of Protein Biomarkers Relevant to Sperm Characteristics and Fertility in Semen in Three Wild Felidae: The Flat-Headed Cat (Prionailurus planiceps), Fishing Cat (Prionailurus viverrinus), and Asiatic Golden Cat (Catopuma temminckii).

Effective wild cat conservation programs with assisted reproductive technologies are being developed in different parts of the world. The flat-headed cat, fishing cat, and Asiatic golden cat are three species among nine wild Felidae in Thailand that are in need of urgent conservation efforts. Here, we assessed routine sperm characteristics and we report the detection of protein biomarkers related to the fertilization process, IZUMO1 and the CRISP family, and apoptotic markers, active or cleaved caspase-3, in semen samples collected from these wild cats. IZUMO1 was located in the equatorial segment of the sperm head, which is the region involved in gamete interaction. The highest levels of IZUMO1 were found in both the sperm pellet and the seminal plasma of the flat-headed cat, as determined by immunoblotting. CRISP2, a sperm-egg fusion assisting protein, and CRISP3 were found in both the sperm pellet and the seminal plasma, and the highest levels were observed in the fishing cat. Positive correlations between certain semen parameters and IZUMO1, CRISP2, and CRISP3 expression were also demonstrated. Cleaved caspase-3 was found in all sperm samples in all three species and was associated with an increase in DNA fragmentation and a decrease in certain semen characteristics such as motility, viability, and intact acrosomes. Our results suggest that the analysis of IZUMO1, the CRISP family, and cleaved caspase-3, along with the routine sperm characteristics, may allow for better success in breeding management in wild Felidae, particularly in the flat-headed cat and the fishing cat.