Acrosomal marker SP-10 (gene name Acrv1) for staging of the cycle of seminiferous epithelium in the stallion

Abstract

The acrosome plays a critical role in sperm-oocyte interactions during fertilization. SP-10 is an acrosomal matrix protein, which is evolutionarily conserved among mammals. The SP-10 antibody has been shown to be useful for staging the seminiferous cycle in the mouse and human. A canonical acrosomal marker; however, has never been used for staging in the horse. The objectives of the present study were to investigate the presence of SP-10 within the horse acrosome using an anti-mouse SP-10 antibody, to classify spermatids based on the shape of the acrosome, and then to use that information to assign stages of the cycle of the seminiferous epithelium. Testes from mature stallions with history of normospermic ejaculates were used for immunohistochemistry. We found that the mouse SP-10 antibody stained the horse acrosome vividly in testis cross-sections, indicating evolutionary conservation. Previous methods based on morphology alone without the aid of an antibody marker showed 8 stages in the horse seminiferous epithelium. Morphological detail of the acrosome afforded by the SP-10 marker identified 16 steps of spermatids. This, in turn, led to the identification of 12 distinct stages in the cycle of the seminiferous epithelium of the horse wherein stage I shows recently formed round spermatids and stage XII includes meiotic divisions; a classification that is consistent with other animal models. The SP-10 antibody marks the acrosome in a way that enables researchers in the field to identify stages of spermatogenesis in the horse easily. In conclusion, we demonstrated that immunolabeling for SP-10 can be an objective approach to stage the cycle of the seminiferous epithelium in normospermic stallions; future studies will determine if SP-10 could be used to assess testicular dysfunction.

1. Introduction

Spermatogenesis is the process by which spermatogonial stem cells, through multiple rounds of divisions and differentiation, eventually produce elongated spermatozoa. Stages of the seminiferous epithelium are characterized by germ cell associations at specific points in mitosis, meiosis, or post-meiotic development, found along the length of the seminiferous tubule [1]. Typically, a cross-section of the seminiferous tubule will show one stage. Germ cells mature collectively by going through a series of stages; consequently, the stages are repeated at regular time intervals at any given space within the tubule [2]. The time it takes for the re-appearance of the same stage at a particular point within the tubule is termed as the cycle of the seminiferous epithelium [3–5].

One way to assess the health of spermatogenesis is to obtain a testicular biopsy and to examine cross-sections of the seminiferous tubules under the microscope. Any abnormalities in cellular associations would be suggestive of an abnormal testicular function and subfertility. Prior knowledge of which cell associations define each stage of the cycle is essential for this histopathological evaluation. The horse industry is a multi-billion dollar industry with a global economic impact. The American Horse Council estimates that the horse industry contributes $122 billion to the U.S economy and impacts 1.7 million jobs. Stallion reproductive disorders, therefore, are important to study as the stakes are enormous where the fertility status of genetically valuable sires can have a major impact on the financial success of a breeding enterprise. Testicular biopsies, obtained using punch biopsies and spring-loaded needle biopsies, have been determined to be safe and diagnostic in stallions [6]. Successful histopathological evaluation of testicular biopsies in cases of infertility such as azoospermia, will depend on the availability of appropriate markers for germ cell development [7–9].

Stage-specific expression and localization of well-characterized spermatogenic markers within the seminiferous epithelium indicate proper progression of spermatogenesis. Markers for germ cell development have not been extensively investigated in the stallion testis compared to the mouse and human. Thus far, a limited number of markers have been demonstrated on the horse. GFRA1, PLZF, and CSFR1 have been shown to be expressed in the early type spermatogonia [10]. Subsequently, UTF1 and Lin 28 were shown as undifferentiated spermatogonial markers [11, 12]. In addition, cKit and Dazl (deleted in azoospermia-like) markers have been shown to be co-stained in differentiating spermatogonial cells [13]. Acrosin binding protein (ACRBP) was shown to be expressed in the spermatocytes, round and elongated spermatids, as well as sperm [14]. Zonadhesin was demonstrated in horse testis cross-sections as well as sperm [15]. In another study, several post-translational modifications of histone H4 were tracked and shown to accompany chromatin remodeling of the sperm nucleus [16]. Importantly, the above studies used antibodies raised against mouse or human sperm proteins, which cross-reacted with the horse. This indicates evolutionary conservation of proteins and pathways involved in male germ cell differentiation.

A canonical acrosomal marker, however, has never been tested in the horse. SP-10 is an acrosomal matrix protein, some of which is retained on the inner acrosomal membrane following the acrosome reaction [17, 18]. We have been studying the evolutionarily conserved acrosomal protein SP-10 in mice and using the gene which encodes SP-10 (Acrv1) as a model to understand testis-specific gene regulatory mechanisms [19–21]. In vitro, function-blocking assays showed that antibodies to SP-10 protein inhibit sperm-zona binding and membrane fusion [22, 23]. Based on these findings, the SP-10 protein was termed as a potential reversible contraceptive vaccine and tested in animal models [24–26]. We have generated high-quality polyclonal antibodies to the mouse SP-10 protein and used it effectively to stage the cycle of the seminiferous cycle in the mouse [19] which compares favorably with that of the periodic acid Schiff’s (PAS) reaction [27]. PAS staining was previously used in the horse [28]. Staging with the SP-10 antibody offers additional advantages over PAS staining such as use in combination with other immunolabelling reagents and immunofluorescence.

Stages of the seminiferous epithelium cycle are well-described for the human, mouse, rat, and dog using the shape of the acrosome as the major criterion. In contrast, staging has been done in the stallion since the 1970s relying mainly on cellular associations [28–33]. Although a majority of the studies reported eight stages of the cycle, at least one report has defined twelve stages in the horse based on the morphology of the acrosome as seen in higher-resolution plastic sections [10]. We reasoned that the SP-10 antibody, well-established for staging of the cycle of the seminiferous epithelium in other speicies [19, 34], might serve well for staining the horse acrosome and resolve the staging issue. The goals of the present study were to a) use the SP-10 antibody to track the development of the equine acrosome during spermatogenesis, and b) use the well-defined acrosomic system to assign stages of the cycle of the seminiferous epithelium in the horse.

2. Materials and methods

2.1. Stallions

All animal procedures conducted in this study were revised and approved by the Institutional Animal Use and Care Committee of the University of Illinois Urbana-Champaign under the protocol (#14200). Three stallions (5 years old Thoroughbred, 8 years old Quarter, and 6 years old Quarter Horse) with a history of normospermia were used in the study. The animals were donated to the University of Illinois Urbana-Champaign to be used in research projects involving semen processing and biotechnology. After the end of the semen studies, the animals were submitted to standard standing castrations.

The animals were sedated with detomidine hydrochloride (0.02 mg/kg, IV) and butorphanol tartrate (0.02 mg/kg, IV). The spermatic cord was blocked with lidocaine hydrochloride (20 mL/cord). Each side of the scrotum was incised, the testis exposed, and the spermatic cord located and gently stretched, before placement of an emasculator for 5 min and ligation of the spermatic cord with absorbable suture (Vycryl, Polyglactin 910, Ethicon, Sommerville NJ). The animals received two doses of flunixin meglumine (1.1 mg/kg, IV, q24h, Butler Schein Animal Health Fort Worth, TX) to control pain and inflammation. In addition, penicillin G procaine (22,000 units/IM q12h, Butler Schein Animal Health) was administered to prevent infection. None of the animals developed any complications post-castration.

2.2. Immunohistochemistry

Immediately after castration, the testes were rinsed in saline solution and transported to the laboratory for histological preparation. Segments from the testes were randomly sampled and placed in Bouin’s solution overnight, before being embedded in paraffin until further processing. The immunohistochemistry (IHC) procedure used for SP-10 staining in horse testes corresponds to that used in mouse [19]. Tissue from Bouin’s fixed testis was processed for paraffin embedding using a Tissue-Tek VIP 1000 processor (Sakura Finetek, Torrance, CA). The samples were sectioned at 4 microns with a Leica RM2125 RTS rotary microtome (Leica Biosystems, Buffalo Grove, IL) and mounted on glass slides. These were deparaffinized with xylene and hydrated through a series of graded ethyl alcohols. IHC pretreatment was performed using citrate buffer pH 6.0 in a vegetable steamer for 60 min. TBS-tween was used as the buffer rinse throughout the staining procedure. Endogenous peroxidase was blocked using 3.0% hydrogen peroxide for 10 min. Nonspecific background blocking was performed using Background Punisher (Biocare Medical, Pacheco, CA) for 20 min. The sections were incubated for 1 hour at room temperature in the primary antibody, guinea pig anti-mouse SP-10 (1:500). The primary antibody was omitted from one section for a negative control. Following rinsing, the sections were incubated in the anti-guinea pig secondary at a 1:200 dilution (Jackson Immuno Research Laboratories, West Grove, PA). DAB (3,3’Diamenobenzidine) (Innovex Biosciences Inc., Richmond, CA) was used as the chromogen with an incubation time of 5 min. Slides were counterstained with hematoxylin, dehydrated, cleared and mounted.

One hundred tubule cross-sections per stallion, at four depths, were randomly selected for evaluation. We used cross-sections that were round so that a transverse, but not longitudinal, section was assessed. Stages were assigned primarily based on the shape and morphology of the acrosome on spermatids revealed by the SP-10 antibody staining, in the spermatids, but the presence or absence of specific types of spermatogonia and spermatocytes were also taken into consideration.

Fixed testes sections from the aforementioned adult stallions were treated with a second marker, γH2Ax (Millipore, now Millipore Sigma, Germany). The antibody was used according to the manufacturer’s supplied instructions for immunocytochemistry, except at a concentration of 1:50. The immunohistochemistry methodology was the same as used with the SP-10 antibody.

2.3. Imaging

Images of the immunostained cross-sections were captured at the Core Facilities at the Carl R. Woese Institute for Genomic Biology, UIUC using the Axiovert 200M Widefield Fluorescence microscope (Zeiss, Pleasanton, CA) with the Apotome Structured Illumination Optical Sectioning System, 40X oil objective, the Axiocam 506 high-resolution camera (Zeiss, Pleasanton, CA), and the Zen Pro software (Zeiss, Pleasanton, CA). Tubules were chosen at random at a low power setting and, once confirmed that tubule sections were appropriately round, the tubule cross-sections were then evaluated with the 40X oil objective. The same software, computer, and settings were used for each image captured. A 50um bar was added to the lower right corner of each image. Once a sizeable number of sections was collected, the cross-sections chosen at random were evaluated as described in the Results section.

3. Results

3.1. Homology between the mouse and horse SP-10 proteins:

The Acrv1 gene codes for the SP-10 protein. We performed a homology search between the mouse and horse SP-10 protein sequences available in the NCBI protein database (NP_031417, and XP_014596630.1, respectively). As shown in Figure 1, there is 61.6% identity within a 206 amino acid region suggesting conservation of the SP-10 protein. The homology was maximum within the carboxyl-terminal part of the SP-10 protein, including the conservation of ten cysteine residues. The mouse and the human SP-10 amino acid sequences also share 60% homology [35]. We have previously reported the generation of a polyclonal antibody against the mouse recombinant SP-10 protein (aa17–261) which works well for demarcation of the acrosome in immunohistochemistry applications [19]. After noting that the horse SP-10 shares extensive homology with the corresponding mouse protein within the aa48–261 region, we reasoned that these antisera would cross-react with the equid SP-10 protein.

Figure 1.

Homology between the horse and mouse SP-10 protein. The aa68 to 261 region of the mouse and aa45 to 247 region of the horse share 61.6% identity. The conserved glutamine and cysteine residues are highlighted.

3.2. SP-10 antibody specific to developing acrosome in stallions

Immunohistochemistry of adult horse testis cross-sections indeed showed that the SP-10 antibody was highly specific to the acrosome. Immunoreactivity was restricted to the acrosome region of round and elongating spermatids, similar to what was observed in the mouse [19]. There was no staining in spermatogonia, spermatocytes, or Sertoli cells within the tubule or with any other cell type in the interstitium (Fig. 2). Thus, the anti-mouse SP-10 polyclonal antibody was deemed suitable as a marker for the acrosome in horse testis cross-sections.

Figure 2.

The SP-10 antibody specifically stains the acrosome of spermatids in horse testis cross-sections. Immunohistochemistry using no primary antibody negative control (A) and anti-mouse SP-10 polyclonal antibody (B) is shown. Note that only round spermatid acrosomes were stained in panel B. No other germ cell type or somatic cells showed immunoreactivity thus indicating the specificity of the SP-10 antibody.

The SP-10 antibody reactivity made it possible to track the development of the acrosome within the round and elongated spermatids (Fig. 3). Based on the distinct morphological patterns of acrosome staining observed, 16 different sequential steps of spermatids were noted in horse testis cross-sections (step1 through step 16 shown in separate panels in Fig. 3). The least mature round spermatids (St 1) showed no antibody reactivity, but in the next stage a round acrosomal granule made its appearance on the nuclear surface (St 2). Two or more of the acrosomal granules coalesced to give rise to a larger vesicle (St 3). The stain uptake representing the acrosome appeared to solidify into a single focus, then the surface of the acrosome resting on the nucleus began to flatten so that the stained acrosomic vesicle took on a more triangular shape (St 4). Once flattened on the nucleus, the acrosome first began to spread to cover half of the nucleus (St 5–6), and then up to two-thirds of the nucleus (St 7–8). The angles formed by the flattened acrosome on the nuclear surface kept progressively increasing in steps 5–8 spermatids. As the round spermatid nucleus continued to mature and change shape, the acrosome conformed to the nucleus as it elongated (St 9). The acrosome began to take on a thinner appearance as it continued to wrap down the sides of the elongating spermatid nucleus (St 10–16). Thus, use of the SP-10 antibody made it easy to visualize the progressive changes in the morphology of the acrosome which in turn led to the identification of 16 steps of spermatid development (St. 1–16, Fig. 3).

Figure 3.

Based on the gradual changes in the morphology of acrosome as revealed by the SP-10 antibody, 16 steps of spermatids were identified in horse testis cross-sections. Step 1 through step 16 spermatids (St1-St16) shown as individual panels from left to right.

3.3. Acrosome development and germ cell associations guide the classification of stages

Morphological changes of the acrosome shown by the SP-10 antibody allowed us to distinguish the cellular associations in a way that was not previously possible in the horse. We were able to assign 12 stages in the cycle of the seminiferous epithelium of the horse based on the preponderance of acrosome staining in tubular cross sections (stages I-XII) (Fig. 4). Steps 1 through 12 spermatids segregated into stages I through XII of the cycle. Steps 13 through 16 elongated spermatids formed the second generation of spermatids within stages I through VIII. The step 16 spermatids at stage VIII were the most mature elongated spermatids ready to be released into the lumen at spermiation. Morphological differences in the nucleus of spermatogonia and spermatocytes were taken into consideration as outlined below when assigning cellular associations to stages (Fig 4).

Figure 4.

The acrosomal marker SP-10 made it possible to classify the horse seminiferous cycle into 12 stages, each stage with a distinct cellular association. Note that steps 1–12 spermatids segregated into stages I through XII of the cycle. Roman numerals at the top of each panel depict stages of the cycle.

In the Stage I seminiferous epithelium, Step 1 spermatids are newly-formed, with no visible acrosomal stain present on the nucleus. Stage I also has Step 13 spermatids, which have elongating nuclei highlighted by the SP-10 antibody stain. At this stage, type A undifferentiated spermatogonia with granular nucleus and little heterochromatin could also be seen in the cross-section (panel A, Fig 4).

Step 2 spermatids, as seen in Stage II, have smaller acrosomal granules forming around the apex of the spermatid nucleus. Stage III tubule epithelium has Step 3 spermatid nuclei highlighted by the more darkly-stained coalesced acrosomal granules. In both stages, elongating spermatids (Step 13–14) bear lighter staining as the acrosome assumes a thinner shape. An abundance of pachytene spermatocytes could be seen near the basement membrane in stages II and III (panels B-C, Fig 4).

In Stage IV, Step 4 round spermatid nuclei have distinct acrosomal caps with a flat surface (panel D, Fig 4). B₁ spermatogonia, with ovoid nucleus and granularity in the nucleoplasm due to heterochromatin, could be seen at stage IV. In Stage V, the acrosomal caps begin to flatten across the nuclear surface taking up about 1/4^th of the surface of the nuclei of Step 5 round spermatids. Step 15 elongating spermatids are seen located deeper in the epithelium. Type B₂ spermatogonia, identified by the round nuclei with increased heterochromatin and granularity, as well as pachytene spermatocytes, could be seen at this stage (panels D-E, Fig 4). Stage VI depicts the acrosome continuing to grow across round spermatid nuclei. Type B₂ spermatogonia and pachytene spermatocytes are present along the basement membrane. At this stage, the Step 15 elongated spermatids begin to migrate towards the lumen of the seminiferous tubules (panel F, Fig 4).

At stage VII, the round spermatids (Step 7) have distinct acrosomes that spread to cover up to half of the nuclei, and the elongated spermatids have matured into Step 16 spermatozoa (panel G, Fig 4). Also at this stage a differentiating A1 spermatogonia cell could be seen. By Stage VIII, the mature Step 16 spermatozoa neatly line the lumen in preparation for spermiation. The Step 8 round spermatid nuclei are covered by dark, defined acrosome caps that cover up to two-thirds of the nucleus. At this stage, most of the step 8 round spermatids are oriented such that their acrosomes point towards the basement membrane (panel H, Fig 4). At stage VIII the first preleptotenes enter meiotic prophase and become leptotene spermatocytes.

Stage IX cross-sections have only one generation of spermatids present because the elongated spermatozoa were released at stage VIII. The Step 9 spermatid nuclei begin to undergo condensation, face the basement membrane, and they begin to lose their round shape. The SP-10 stain covers nearly all the step 9 spermatid nucleus, except for the caudal aspect that is elongating. Leptotene and pachytene spermatocytes are seen at stage IX near the basement membrane (panel I, Fig 4).

Stage X cross-sections have Step 10 spermatids with nuclei that no longer appear round, but rather take on a teardrop shape, with SP-10 staining covering the apical aspect of the nucleus that is facing the basement membrane. Stage XI cross-sections display Step 11 nuclei that are beginning to take on an elongated shape as the distinctly marked acrosomes begin to thin and flatten around the nuclei. Differentiating type A₂ spermatogonia, which possess the largest nuclear volume among spermatotogonia [10], could be seen at stage XI. Zygotene spermatocytes are found between the pachytene spermatocytes and the basement membrane in these stages (panels J-K, Fig 4).

Stage XII is typified by the characteristic appearance of pachytene spermatocytes in meiotic divisions (I and II) with an equatorial arrangement of chromosomes. Secondary spermatocytes, between the two divisions, are rarely observed but when present have a nucleus that is larger than the step 1 round spermatids, but smaller than the pachytene spermatocytes. Step 12 elongating spermatids, darkly stained by the acrosomal antibody, could be seen in bundles along the luminal surface (panel L, Fig 4).

Overall, immunolabelling of the acrosome greatly aided in the identification of 12 distinct stages in the cycle of seminiferous epithelium of the horse.

3.4. Multiple but contiguous stages per cross-section

While evaluating the horse testis cross-sections, we observed that in general, a single cross-section often showed more than one stage of the seminiferous cycle. As illustrated in Fig 5, panels A-D, separate patches (denoted by the dashed line) could be seen representing different stages of the cycle. Panel A shows stages I, II, and III. Stage I is characterized by round spermatids lacking SP-10 staining, whereas the antibody reactivity becomes stronger in II and III. Panel B shows patches of stages IV and V; the cap-shaped acrosome of step 4 and slightly more spread out acrosome of step 5 spermatids could be seen in zones separated by the dashed lines. Panel C shows stages IX, X, and XI, characterized by the typical step 9–11 spermatid acrosome staining as described in the section above (Fig 3). Panel D shows stages XII and I; the anaphase chromosomes forming meiotic figures being the characteristic feature of stage XII and step 1 spermatids lacking SP-10 staining representing stage I. A total of 300 tubule cross-sections were staged from 3 separate horses; 72% displayed more than one stage per cross-section. Per horse, less than 30% of tubule cross-sections represented a single stage (Fig. 6). It is interesting to note that where multiple stages were present within the same cross-section, the stages represented a continuum. Previous literature has acknowledged the presence of multiple, perhaps transition stages, yet the availability of a definite acrosomal marker (SP-10) in the present study made it possible to determine the frequency of such appearance [36].

Figure 5.

The presence of multiple but adjacent stages of the seminiferous cycle in horse testis cross-sections. Panels A-D were chosen to show a representation of various stages between 1 through 12. A, type A spermatogonium; B, type B spermatogonium; P, pachytene spermatocyte, S, Sertoli cell; Z, zygotene spermatocyte; MF, meiotic figure.

Figure 6.

Percentage of testis cross-sections showing more than one or one stage of the seminiferous cycle. A total of 300 cross-sections were counted from 3 separate horses (100 from each horse).

3.5. Spermatocyte marker γH2AX staining confirms multiple stages per cross-section

We sought to use a different germ cell marker, a spermatocyte-staining antibody, in order to confirm the presence of multiple stages within a single cross-section. Using a commercial antibody to γH2AX, with the aforementioned immunohistochemical techniques, we appreciated a similarly heterogeneous appearance in the staining pattern of tubule cross-sections (Fig. 7). The anti-γH2AX marker highlights double-strand breaks (DSBs) in DNA, which occur during meiosis [37]. As spermatocytes undergo meiosis, DSBs are exposed, recruiting the phosphorylated form of histone H2AX (γH2AX) foci to the sites. As a result, the leptotene, zygotene, and pachytene spermatocytes pick up the γH2AX antibody stain. In the representative images shown (panels B-C, Fig 7), it can be appreciated that leptotene and zygotene spermatocytes at stages X-XI showed dark staining whereas pachytene spermatocytes at stages VII-VIII showed lighter staining. This is consistent with a previous report of γH2AX staining of meiotic cells of the stallion testis [38]. The other germ cells of the cross-section are devoid of reactivity. Based on spermatocyte staining with the γH2AX antibody in panels B and C of Fig 7, patches representing different stages of the cycle could be identified, thus confirming multiple stages within a single cross-section of the horse testis. The frequency of occurrence of multiple stages within a cross-section was similar to that observed with the SP-10 antibody (data not presented). Thus, γH2AX staining supported the observation that multiple but contiguous stages were present in the seminiferous tubule cross-sections.

Figure 7.

Confirmation of the presence of more than one stage per cross-section using a second marker, anti-gamma H2AX antibody. Panel A: negative control; Panels B and C: note the gamma H2AX immunoreactivity change in different parts of the same cross-section, similar to what was observed with the SP-10 antibody in Figure 5.

4. Discussion

Immunolabelling with the SP-10 antibody clearly demarcated the acrosome in horse testis cross-sections. Based on the visualization of progressive changes in the morphology of the acrosome, 16 different types of spermatids (steps 1–16) and 12 distinct stages of the cycle of the seminiferous epithelium were identified. Stage I of the cycle included nascent post-meiotic round spermatids, spermiation occurred at stage VIII, and stage XII represented spermatocytes undergoing meiotic divisions.

The first report of stages in the horse was by Swierstra et al. in 1974, who identified 8 different types of cellular associations in testis cross-sections and therefore stated that there are 8 stages in the cycle of the seminiferous epithelium [29]. This view was further endorsed by a group using three additional methods of visualizing stages: a) transillumination observation of enzymatically digested seminiferous tubules, b) Nomarsky optics to observe sectioned testis as well as whole mounts, and c) periodic acid-Schiff reaction (PAS) for carbohydrate macromolecules in the acrosome [28, 31, 32]. Using all three methods, 8 stages in the cycle were proposed. Two of these studies pre-dated the availability of useful immunological markers for male germ cells which would have further separated the cellular associations with finer resolution. In rodents, PAS staining helped to differentiate up to 14 stages [1, 27]. Using PAS staining, however, Heninger et al [28] demonstrated 8 stages of the seminiferous cycle in the horse similar to what was observed in other large mammals [39, 40]. The intensity of PAS staining can be inconsistent depending on fixation, embedding and thickness of sections; which may help to explain prior labeling of only 8 stages in the horse testis.

In a review article, Amann pointed out that following the changes in the morphology of the developing spermatids might be an alternative means of classification as had been done by then in rat and bull [36]. Amann predicted that the above method might show 10–14 cellular associations. The present study, using the evolutionarily conserved acrosomal marker SP-10 to label the developing spermatids, along with the recognition of morphological changes, indeed showed 12 distinct cellular associations in the horse. It is interesting to note that using antibodies to another well-characterized acrosomal protein, acrosin, Muciassia et al., identified 12 stages in the human seminiferous cycle and suggested that the six-stage classification be revised [41]. The SP-10 antibody used in the present study greatly improved the ease and efficiency of staging stallion seminiferous tubule cross-sections due to the specificity and localization of acrosome vesicles. Using this marker allowed for a clear discernment of the development of the sperm acrosome, and, by extension, the spermatid itself. Prior to our study, Costa and collaborators used higher-resolution plastic sections to follow the development of the acrosomic system (without the aid of an immunological marker), and were also able to distinguish 12 stages in the stallion [10].

The 8-stage classification model begins with the spermiation as stage I and places meiosis at stage IV, in the middle of the cycle [29, 32]. In the 12 stage classification, meiosis takes place in stage XII, and the post-meiotic spermatid development begins with stage I, which has been well-established for several animal models [5]. One other study compared nucleoprotein exchange events in murine and equine spermatogenesis and attempted to revise the stallion stages to resemble the mouse [16]. They placed the first post-meiotic round spermatids within stage I and ended with meiotic divisions in the final stage (stage VIII) but still grouped the cellular associations into only 8 stages. Ketchum et al., acknowledged that using the acrosome system as a guiding marker would lead to 12 stages but argued that in instances where acrosomal marker usage is not feasible, the 8-stage system should be applied [16]. We propose that the canonical acrosome marker SP-10 should be easy to apply for staging the horse spermatogenic cycle to bring it more uniformity with the widely used mouse system. Our laboratory routinely supplies the SP-10 antibody as a gift to investigators for use as a marker for the acrosome and spermatids [42–47]. In addition, several vendors offer SP-10 antibodies commercially but their usefulness in staging has yet to be determined.

The present study using the SP-10 antibody established that two-thirds of horse testis cross-sections show more than one stage of the cycle represented. We confirmed this observation by using a spermatocyte marker γH2AX which also showed a similar trend. Where more than one stage was identified, the stages tended to be in continuum. The presence of multiple stages within a single cross-section and the concept of transition has been reported [28, 48]. In fact, careful observation of images from previously published manuscripts revealed the presence of multiple stages within a cross-section. Fig 3 of Kim et al.’s paper showing ACRBP immunofluorescence staining of horse testis cross-sections is a case in point [14], where entire cross-section of the testis make it possible to see multiple stages. The panels representing stages III, IV, V, VI, and VIII show ACRBP immunofluorescence in patches within the cross-section, thus representing more than one stage [14]. In the same vein, in Fig 2 of Ketchum et al, the authors labeled multiple but contiguous stages in panels A and B stained by the Histone H4 acetylation (penta) antibody [16].

In conclusion, the present study demonstrated that the evolutionarily conserved acrosomal protein SP-10 is present in the equids. The SP-10 antibody staining provided fine resolution to the shape of the developing acrosome of the horse, leading to the identification of 12 stages of the cycle of the seminiferous epithelium. Future studies are warranted to explore the usefulness of this marker for the characterization and diagnosis of testicular dysfunction in stallions.

Highlights.

1. The acrosomal protein SP-10 plays a critical role in sperm oocyte interaction during fertilization.

2. SP-10 antibody has been successfully used in the mouse and human to stage the cycle of seminiferous epithelium

3. This is the first report to our knowledge that used a canonical acrosomal marker (SP-10) to stage the cycle of seminiferous epithelium in the horse

4. Clear demarcation of the acrosome by the SP-10 antibody helped identify 12 stages in the cycle of seminiferous epithelium of the horse providing a refinement to the historical 8-stage classification