Postacrosomal Area Research Articles

P-038 Sperm head morphometric reference values of 1856 sperms in fertile Asian males

Abstract Study question To explore comprehensive normal sperm morphometric reference values by measuring 1856 sperms in fertile males using artificial intelligence (AI) technique. Summary answer Four novel describing parameters of sperm head (size, shape, area, and uniformity) and 14 other features for normal sperm head were extracted and characterized. What is known already Sperm head morphology is crucial for male factor infertility diagnosis and assessment of male reproductive potential. The human spermatozoon is one of the most diverse cell types known. However, most assessment criteria have only 3 morphometric parameters (Length, width, and length/width ratio) for sperm head morphometric measurement. We detailed the features of sperm head by defining and characterizing 14 parameters based on AI. Study design, size, duration This is a prospective observational study. The study included 1856 normal sperm images from 23152 stained sperm acquired by an Automated Sperm Morphology Analysis (ASMA) system. The measured sperm morphometry data were acquired from May 2020 to December 2021. Participants/materials, setting, methods All participants (54 fertile males) had fathered a child naturally during the past year. The measured sperm morphometry data were acquired from candidate donors of the Sichuan Province Human Sperm Bank (SPHSB, Chengdu, China). Images of 23,152 stained sperm captured by ASMA. All sperm images were assessed by three experienced technicians. Finally, we concluded 14 morphometric data from normal sperm by measuring sizes in pixels. Main results and the role of chance (1) Characteristics of sperm head size and shape: We present eight sperm head size and shape parameters for the 1856 visually normal spermatozoa. The median head size parameters included head length of 4.02 (95% CI: 4.01–4.05) μm, head width of 2.52 (95% CI: 2.52–2.54) μm, length to width ratio of 1.59 (95% CI: 1.59–1.62), and head girth of 11.18 (95% CI: 11.17–11.25) μm. The median head symmetry parameters included short axis symmetry (SAS) of 92.1% (95% CI: 88.3%–89.2%) and the long axis symmetry (LAS) of 94.3% (95% CI: 91.6%–92.3%). The median head shape parameters included median of ellipse intersection over union (EIoU) of 88.9% (95% CI: 85.4%–86.3%) and girth intersection over union (GIoU) of 102.4% (95% CI: 102.4%–102.5%). (2) Characteristics of sperm head area and acrosome uniformity: Descriptive analysis of the sperm head area characteristics included a median sperm head area of 7.72 (95% CI: 7.74–7.85) μm2, acrosome area of 3.67 (95% CI: 3.77–3.88) μm2, acrosome area ratio of 48.2% (95% CI: 47.8%–48.6%), post-acrosomal area of 3.95 (95% CI: 3.95–4.00) μm2, acrosome uniformity of 88.6% (95% CI: 88.6%–89.1%), and nuclear uniformity of 100.0% (95% CI: 99.8%–99.9%). Limitations, reasons for caution This study had several limitations that should be noted. The use of samples from a single center, covering relatively young participants from only seven provinces in China, could limit the usage of our findings. The use of only three technicians to assess the samples may have introduced a subjective bias. Wider implications of the findings The measurement technique was accurate, objective, and highly informative by AI. The data provide extremely detailed information for reference studies and comparison of sperm head parameters among different male groups. These new parameters can be applied in routine semen analysis when assessing sperm morphology and male fertility potential. Trial registration number not applicable

MP38-17 THE PROFILE OF HUMAN SPERM OXYSTEROLS IN NORMAL AND SUBFERTILE PATIENTS: RESULTS FROM A PROSPECTIVE MULTICENTRE STUDY

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology (MP38)1 Apr 2020MP38-17 THE PROFILE OF HUMAN SPERM OXYSTEROLS IN NORMAL AND SUBFERTILE PATIENTS: RESULTS FROM A PROSPECTIVE MULTICENTRE STUDY Antonio Luigi Pastore*, Andrea Fuschi, Yazan Al Salhi, Gennaro Velotti, Lorenzo Capone, Alessia Martoccia, Pietro Paolo Suraci, Chiara Zerbinati, Luigi Iuliano, and Antonio Carbone Antonio Luigi Pastore*Antonio Luigi Pastore* More articles by this author , Andrea FuschiAndrea Fuschi More articles by this author , Yazan Al SalhiYazan Al Salhi More articles by this author , Gennaro VelottiGennaro Velotti More articles by this author , Lorenzo CaponeLorenzo Capone More articles by this author , Alessia MartocciaAlessia Martoccia More articles by this author , Pietro Paolo SuraciPietro Paolo Suraci More articles by this author , Chiara ZerbinatiChiara Zerbinati More articles by this author , Luigi IulianoLuigi Iuliano More articles by this author , and Antonio CarboneAntonio Carbone More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000887.017AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Cholesterol is the main lipid component of sperm cell that is essential for sperm membrane fluidity, capacitation, and acrosomal reaction. Recent data obtained in bovine sperm showed that sperm capacitation is associated with the formation of oxysterols, oxidized products of cholesterol. The main objective of the present study was to identify and quantify, for the first time, the different species of oxysterols in human semen from normozoospermic, oligoasthenoteratozoospermic and asthenozoospermic patients. The secondary aim was to investigate the potential role of oxysterols in sperm pathophysiology. METHODS: To investigate the profile of human sperm oxysterols in subjects with normal and altered sperm characteristics, we recruited 150 consecutive subjects referring at the Centre of Andrology of Department of Urology for analysis of seminal fluid between March 2017 and June 2018. In order to investigate the possible correlation to oxidative stress and semen oxysterols profile, we included a group of patients with varicocele given the demonstrated relationship between varicocele and oxidative stress. Semen analysis was assessed by light microscope according to World Health Organization guidelines (WHO, 2010). Oxysterols were determined by GC–MS using deuterium-labelled internal standards as described by Dzeletovic et al. RESULTS: Complete oxysterol profile was obtained in 134 human semen of normozoospermic, oligoasthenoteratozoospermic, asthenozoospermic and varicocele patients. Oxysterols analyzed included seven autoxidation- and five enzymatically-generated oxysterols. Among the 12 oxysterols analyzed, 25-hydroxycholesterol (25-HC) resulted the most abundant oxysterol in normozoospermic subjects, and turned out to be the only one that differed significantly (p<0.0001) among the 4 groups. It was higher in the normozoospermic group (21.63±18.47 ng/mL, mean±SD) than oligoasthenoteratozoospermic (2.59±2.93, ng/mL, mean±SD), asthenozoospermic (5.59±3.17) and varicocele (13.48±11.81, ng/mL, mean±SD) groups. Furthermore, 25-HC positively correlated with the spermatozoa number (r=0.72, p<0.0001). CONCLUSIONS: In conclusion, to the best of our knowledge, this is the first study providing evidence for the feasibility of detection and quantification of oxysterols in human semen samples. We found in spermatozoa the presence of cholesterol 25-hydroxylase and its preferential accumulation in the neck and the post acrosomal area. 25-HC was able to induce calcium ions and cholesterol depletion in the acrosomal region, in an extent comparable to that of a capacitating medium. This finding offers the opportunity to identify a new molecular player involved in pre-fertilization processes. Source of Funding: None © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e575-e576 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Antonio Luigi Pastore* More articles by this author Andrea Fuschi More articles by this author Yazan Al Salhi More articles by this author Gennaro Velotti More articles by this author Lorenzo Capone More articles by this author Alessia Martoccia More articles by this author Pietro Paolo Suraci More articles by this author Chiara Zerbinati More articles by this author Luigi Iuliano More articles by this author Antonio Carbone More articles by this author Expand All Advertisement PDF downloadLoading ...

The proportion of tyrosine phosphorylated spermatozoa in cryopreserved semen is negatively related to crossbred bull fertility

Sub-fertility is a major problem in crossbred bulls. Identification of subtle differences in the quality of cryopreserved spermatozoa among bulls belonging to different fertility rankings would help determine the latent fertility of semen before their use at field conditions. In the present study, we assessed the status of tyrosine phosphorylation, membrane integrity and acrosome reaction of cryopreserved spermatozoa in crossbred bulls (n = 22) with different levels of field fertility and assessed their relationship with fertility. Bulls were categorized into above-average (n = 4), average (n = 14) and below-average (n = 4) based on their different field fertility rates. The progressive sperm motility was significantly (P < 0.05) higher in above-average fertile bulls compared to either average or below-average fertile bulls whereas sperm membrane integrity and acrosomal reaction status did not differ among the three groups. The proportion of live tyrosine-phosphorylated spermatozoa were significantly (P < 0.05) higher in below-average and average fertile bulls compared to above-average bulls. Immunolocalization of protein tyrosine phosphorylation in spermatozoa revealed that the proportion of spermatozoa showing tyrosine phosphorylation at acrosome and post-acrosomal area (APA) and at acrosome, post-acrosome and tail (APAT) were significantly (P < 0.05) higher in below-average fertile bulls than other groups. The APA pattern (r = −0.605; P < 0.01) and APAT (r = 0.507; P < 0.05) pattern were significantly and negatively correlated with bull fertility. It was concluded that the proportion of live tyrosine-phosphorylated spermatozoa in cryopreserved semen was negatively related to bull fertility.

Mass spectrometry profiling of oxysterols in human sperm identifies 25-hydroxycholesterol as a marker of sperm function

Cholesterol is a main lipid component of sperm cell that is essential for sperm membrane fluidity, capacitation, and acrosomal reaction. Recent data obtained in bovine sperm showed that sperm capacitation is associated to the formation of oxysterols, oxidized products of cholesterol. The aim of this study was to profile oxysterol content in human semen, and to investigate their potential role in sperm pathophysiology. Among the 12 oxysterols analyzed, 25-hydroxycholesterol (25-HC) resulted the most represented in normozoospermic samples, and its concentration positively correlated with spermatozoa number. We detected Cholesterol 25-hydroxylase, the enzyme responsible for 25-HC production, in human spermatozoa at the level of the neck and the post acrosomal area. Upon incubation with spermatozoa, 25-HC induced calcium and cholesterol transients in connection with the acrosomal reaction. Our results support a role for 25-HC in sperm function.

Measurement of activity and concentration of paraoxonase 1 (PON-1) in seminal plasma and identification of PON-2 in the sperm of boar ejaculates.

This study revealed and characterised the presence of the antioxidant enzymes paraoxonase (PON) type 1 (PON-1, extracellular) and type 2 (PON-2, intracellular) in boar semen. To evaluate PON-1, an entire ejaculate from each of ten boars was collected and the seminal plasma was harvested after double centrifugation (1,500g for 10 min). Seminal plasma was analysed for concentration as well as enzymatic activity of PON-1 and total cholesterol levels. Seminal-plasma PON-1 concentration ranged from 0.961 to 1.670 ng/ml while its enzymatic activity ranged from 0.056 to 0.400 IU/ml, which represent individual variance. Seminal-plasma PON-1 concentration and enzymatic activity were negatively correlated (r = -0.763; P < 0.01). The activity of seminal-plasma PON-1 negatively correlated with ejaculate volume (r = -0.726, P < 0.05), but positively correlated with sperm concentration (r = 0.654, P < 0.05). Total seminal-plasma cholesterol concentration positively correlated with PON-1 activity (r = 0.773; P < 0.01), but negatively correlated with PON-1 concentration (r = -0.709; P < 0.05). The presence of intracellular PON-2 was determined via immunocytochemistry in spermatozoa derived from artificial insemination. PON-2 localised to the post-acrosomal area of the sperm head and principal piece of the tail in membrane-intact spermatozoa. In summary, PON is present in boar semen, with PON-1 at low levels in seminal plasma and PON-2 within the spermatozoa. Further studies are needed to characterise the relationship between antioxidant PONs with sperm and other seminal-plasma parameters.

Characterisation of Lyzls in mice and antibacterial properties of human LYZL6

C-type lysozyme genes (Lyzls) belong to the class of lysozymes and are highly expressed in the testis and epididymis. The members Lyzl4 and Spaca3 have been reported to play a role in sperm-egg binding and fertilisation in mice. However, the function of the remaining two mouse c-type lysozyme genes, Lyzl1 and Lyzl6, is still not clear. In the present study, we analysed the tissue expression and androgen-dependent expression of mouse c-type lysozyme genes and the possible contribution of human recombinant LYZL6 (rLYZL6) to immunity. The expression of Lyzls was detected by RT-PCR, Western blots, immunohistochemistry and immunofluorescence. The bacteriolytic activity of rLYZL6 was analysed by a colony-forming assay. In mice, the expression of Lyzl genes was mainly in the testis and epididymis in a developmentally regulated manner and androgen- or testicular factor-regulated manner. Immunodetection revealed the presence of LYZL6 protein in primary spermatocytes and round spermatids of the testis and on the post-acrosomal area and midpiece of mature epididymal spermatozoa. The rLYZL6 protein exhibited antibacterial activity. From the results, Lyzls may play a role in mitochondrial function of spermatozoa and LYZL6 may contribute to the innate immunity of the male genital tract.

Changes in Actin Distribution of Ram Spermatozoa under Different Experimental Conditions

The purpose of this study was to determine the presence of actin in ejaculated ram spermatozoa and the changes of localization that actin undergoes as a consequence of certain in vitro-induced physiological states. Using indirect immunofluorescence (IIF), three different patterns of staining (defined immunotypes) were established in ejaculated sperm. The three sperm immunotypes showed actin labelling in flagellum, neck and post-acrosomal area, differing on the labelling in the acrosomal region that was complete in immunotype 1, partial (frequently concentrated in the apical area, punctuate form) in immunotype 2, and totally absent in immunotype 3. The main subpopulation in ejaculate was immunotype 1 that represented 68% of total sperm, while 21% corresponded to immunotype 2 and only 10% corresponded to immunotype 3. Selection of high-quality sperm using a dextran/swim-up procedure hardly influenced the proportion of each immunotype resulting in a slight increase in type 1 sperm. Cold-shock treatment and in vitro capacitation induced a partial loss of actin labelling in the acrosomal area, whereas the ionophore-induced acrosomal exocytosis provoked a total loss of the acrosomal actin labelling, a phenomenon partially inhibited by phalloidin.

Immunolocalization and Possible Functional Role of PSP‐I/PSP‐II Heterodimer in Highly Extended Boar Spermatozoa

PSP-I/PSP-II heterodimer is a major protein of boar seminal plasma which is able to preserve, in vitro, the viability, motility, and mitochondrial activity of highly extended boar spermatozoa for at least 5 hours. However, little is known about the binding pattern of the heterodimer to the sperm plasma membrane and its eventual relation with the maintenance of the sperm functionality. The present study investigated the effect of exposing highly extended boar spermatozoa (1 million/mL) to 1.5 mg/mL of PSP-I/PSP-II for 0.5, 5, and 10 hours at 38 degrees C on sperm characteristics and the changes in PSP-I/PSP-II localization as a result of both the addition of PSP-I/PSP-II to the extender and the incubation time. Exposure of the spermatozoa to PSP-I/PSP-II preserved sperm viability, motility, and mitochondrial activity when compared to nonexposed spermatozoa. This protective effect lasted for 10 hours (P < .05). After immunolabeling of highly extended semen with rabbit monospecific polyclonal antibody against PSP-I/PSP-II, the percentage of immunopositive spermatozoa declines over time from 71% (0.5 hours) to 49% (10 hours). However, more than 80% of spermatozoa remained labeled during the 10-hour incubation period if PSP-I/PSP-II was added. Scanning electron microscopy revealed 4 different binding patterns. The heterodimer was mainly localized to the acrosomal area, being redistributed to the postacrosomal area or lost during in vitro incubation. In conclusion, the protective effect of the heterodimer appears to be related to its adhesion to the acrosomal area, and the loss of this protective effect coincides with a stepwise redistribution of PSP-I/PSP-II during incubation.

Characterization of galactose-binding proteins in equine testis and spermatozoa

Carbohydrate-binding proteins are thought to be involved in a myriad of sperm functions including sperm–oviductal and sperm–zona interactions. Recent studies in our laboratory have characterized galactose-binding proteins on equine spermatozoa as possible candidate molecules for sperm adhesion to oviduct epithelial cells. In the current study, equine sperm membrane proteins were subjected to galactose-affinity chromatography, and bound proteins were eluted with excess galactose in a calcium-free buffer. The eluted fraction recovered after galactose-affinity chromatography was used for generation of a polyclonal antibody which was immobilized on an affinity column to recover a purified protein from equine sperm extracts. Several protein bands of approximately 70, 25, and 20–18 kDa were detected with a major band at 25 kDa on immunoblots which was subjected to N-terminal amino acid sequencing. These galactose binding proteins (GBP) were specific to sperm and testis and were absent in all the somatic tissues tested. Based upon immunocytochemistry, GBP were localized over the sperm head. In noncapacitated sperm, fluorescent labeling was observed over the rostral sperm head as well as the postacrosomal area; whereas in capacitated sperm, the labeling was localized primarily in the equatorial segment. Immunohistochemistry of equine testis demonstrated abundant staining in the adluminal region of the seminiferous tubules corresponding to round spermatids. In summary, this study demonstrates the presence of testis- and sperm-specific galactose binding proteins in the horse. The function of these proteins remains to be determined.

Sperm antigen MSH27 participates in sperm-egg membrane fusion

A monoclonal antibody (mAb), MSH27 has been selected from an mAb library which has been prepared with separated mouse sperm heads as antigens. Three aspects of evidence indicated that the MSH27 antigen was involved in the process of sperm-egg membrane fusion. After the acrosome reaction, the antigen was located at sperm equatorial segment and in postacrosomal area, where, it was widely accepted, the sperm-egg membrane fusion initially occurred. Inin vitro fertilization, the MSH27 antibody could decrease the index of sperm-egg membrane fusion, but made no effects on sperm approaching and binding to the plasma membrane of eggs. The inhibition showed an antibody concentration-dependent manner, with a rate decrease of 90% at 600 μg/mL immuno-globulin IgM. Furthermore, the antigen was able to affect the sperm-egg membrane fusion directly. The fusion index was obviously reduced after the zona-free eggs were exposed to the antigen purified by immuno-affinity chromatography. These, all together, demonstrated that the MSH27 antigen played an important role in spermegg membrane fusion.

Antigen changes of monoclonal antibody MSH27 in process of post-testicular maturation (in mice)

An anti-mouse spermatozoon monoclonal antibody, MSH27, as well as its purified antigen, can block sperm-egg membrane fusion. As a candidate protein for sperm-egg membrane fusion, the sperm antigen was investigated in the process of post-testicular maturation (PTM). The molecule was produced in testes and located on the plasma membrane of the postacrosomal area of the spermatozoon. However, the epitope recognized by the MSH27 (MSH27Ep) was not exposed until the occurrence of the acrosome reaction. In the process of fertilization, spermatozoa must complete the acrosome reaction before penetrating across the zona pellucidas (ZPs) to approach the plasma membrane of eggs. The effects of the acrosome reaction and penetration of the ZP on the exposure of the MSH27Ep were also studied. It was shown that the percentage of the spermatozoa with the MSH27Ep exposed increased followed with their mature status in PTM. In fact, it had a linear correlativity with the rate of the acrosome reaction. After spermatozoa had passed ZPs, almost all of them became the MSH27-positive in immuno-staining compared with only a part of the spermatozoa after the acrosome reaction. In a word, the exposure of MSH27Ep was coincident with attaining the ability to penetrate the plasma membrane of eggs.

Changes in binding of lectins to epididymal, ejaculated, and capacitated spermatozoa of the rhesus monkey.

The present study was undertaken to evaluate the changes in rhesus monkey sperm surface glycoconjugates during maturation, ejaculation, and capacitation in order to provide background information that would help in evaluating adverse effects, if any, caused by the use of contraceptive agents. Adult sexually mature rhesus monkeys were castrated under ketamine anaesthesia. Percoll purified sperm from different epididymal segments and motile-ejaculated spermatozoa prior to and following in vitro capacitation were exposed to FITC-labeled lectins. Live or sperm prefixed with paraformaldehyde/alcohol were used. Quantitative analysis of changes in lectin binding was done immunoassay. The majority of live spermatozoa did not show binding of Con A and PNA, whereas uniform labeling of WGA to sperm from corpus epididymidis onward was seen. Unfixed spermatozoa showed marked variation in the pattern of lectin binding. The majority of spermatozoa fixed with paraformaldehyde or alcohol showed maximum lectin labeling over the acrosome, but the postacrosomal area invariably did not bind the lectins. Major changes in the localization of Con A, PNA, or WGA to prefixed spermatozoa were not seen during epididymal transit and in the ejaculate. In capacitated acrosome-reacted spermatozoa, Con A, PNA, and WGA were localized mainly in the equatorial segment. Binding of Con A and PNA was abolished by using lectins preincubated with appropriate inhibitor saccharides. Sperm, exposed to FITC-WGA preincubated with inhibitor sugar, did not show complete inhibition. Quantitative analysis of lectin binding by immunoassay showed increase in binding of lectins during epididymal transit with maximum binding in sperm from the corpus epididymidis. The variations in lectin labeling of live spermatozoa could be due to redistribution of sperm surface sugars or membrane damage. The changes in lectin labeling during maturation and capacitation may be associated with their role in ovum recognition and fusion.

Actin, α‐actinin, and spectrin with specific associations with the postacrosomal and acrosomal domains of bovine spermatozoa

Characteristic membrane changes in spermatozoa culminating in acrosome reaction and sperm-egg fusion, and suspected involvement of actin-containing cytoskeleton in membrane changes in general, prompted us to investigate subcellular distribution of actin and actin-binding proteins in bovine spermatozoa subjected to various extractions which sequentially denude the sperm investments. Spermatozoa were treated with either 1% SDS, 0.1% Triton X-100, 0.1% Hyamine, or 1 M MgCl2 or were sonicated. Immunostaining of actin, alpha-actinin, spectrin, and acrosin as well as electron microscopic analysis of extracted spermatozoa were carried out. Extractions caused evagination of the acrosomal lamina which retained focal contacts with the inner acrosomal membrane. Extractions further revealed lateral prongs at the anterior border of the postacrosomal sheath. Labeling for alpha-actinin and spectrin was localized in the acrosin-positive acrosomal lamina, neck, and principal piece, the latter containing also relatively extraction-resistant oligomeric or polymerized actin. In the postacrosomal area, actin was accumulated in the extraction-resistant posterior ring structure and anteriorly at the sites apparently related to the lateral prongs. Notably, spectrin reactivity was enhanced by MgCl2 in head, neck, and principal piece, and sonication abolished cytoskeletal immuno-reactivity in the head. Destabilization of membranes with selected extractions induces changes in the acrosomal lamina mimicking acrosomal vesicle formation. The lateral prongs and posterior ring structure, respectively, may serve as anterior and posterior anchors for the extraction-resistant post-acrosomal sheath. The lateral prongs may also be a merger zone for actin, alpha-actinin, and spectrin with important implication on sperm function. The latter two proteins may be involved in acrosomal vesicle formation. It is apparent that extractions have a significant effect on the detectability of sperm cytoskeletal elements.

Immunocytochemical detection of actin and 53 kDa polypeptide in the epididymal spermatozoa of rat and mouse.

Presence of immunocytochemically detectable actin in the rat and mouse sperm head has been enigmatic for years. In this study, we demonstrate actin in the perinuclear theca and show that the detection of actin epitopes in the rat and mouse epididymal spermatozoa can effectively be enhanced by pre-extraction of sperm cells with SDS. The study with one monoclonal and one polyclonal anti-actin antibody was carried out at conventional and confocal fluorescence and electron microscope level, and by immunoblotting of proteins isolated from the head and tail fractions. In the head of the control methanol-acetone fixed rat spermatozoa, the polyclonal antibody gave a stronger immunostaining in the postacrosomal area and in the perforatorium than the monoclonal antibody. In the mouse sperm head, the monoclonal antibody labeled the ventral edge of the postacrosomal area and slightly the perforatorium, whereas the polyclonal antibody stained the entire perinuclear space. In the SDS-extracted spermatozoa, an intense postacrosomal and perforatorial labeling was obtained with both antibodies but, in particular in the rat spermatozoa, the middle lateral portion of the postacrosomal segment remained unlabeled. Sonication seemed to cause structural modifications which specifically impeded staining with the monoclonal antibody. Both antibodies detected actin in the basal plate and the monoclonal antibody in the neck. Amorphous matrix of the connecting piece showed immunogold labeling. In the tail, the monoclonal antibody recognized actin and a relatively basic 53 kDa polypeptide, whereas the polyclonal antibody reacted with several protein bands. SDS-soluble actin of the tail was addressed to the midpiece and the SDS-insoluble 53 kDa protein profoundly to the outer dense fibers of the principal piece. Intense labeling of actin in the SDS-extracted rat and mouse spermatozoa was presumably due to the generated demasking of actin epitopes embedded in the perinuclear cytoplasm. The results are important in confirming that actin in the rat and mouse sperm head is not lost during spermiogenesis but apparently contributes to the three-dimensional packing of the mature perinuclear cytoplasm. This study further demonstrates the importance of the methods used in sample preparation and advantages of confocal microscopy when attempting to detect cytoskeletal proteins which, as in spermatozoa, may occur in small quantities.

Immunolocalization of proacrosin/acrosin in rabbit sperm during acrosome reaction and in spermatozoa recovered from the perivitelline space

The participation of acrosin in mammalian sperm penetration through the zona pellucida has been amply debated. In this paper we report the immunolocalization--by silver enhanced immunogold technique using ACRO-8C10 monoclonal antibody to human acrosin--of proacrosin/acrosin on ejaculated rabbit spermatozoa incubated in vitro in a capacitating medium and on spermatozoa recovered from the perivitelline space. After incubation in a capacitating medium, four different patterns were observed: (1) no labeling on acrosome intact spermatozoa; (2) labeling on the rim of the head; (3) labeling on the whole acrosome area; and (4) no labeling on acrosome reacted spermatozoa. At the start of incubation, spermatozoa with pattern 1 were the most abundant, whereas at the end of the 32 h incubation period, patterns 2 and 3 were the most frequent. On the other hand, 625 perivitelline spermatozoa were recovered from 17 fertilized rabbit eggs, of which 26% were labeled with the antiacrosin monoclonal antibody ACRO-8C10 in two different areas: (1) only on the equatorial region; and (2) only on the postacrosomal area. These results are consistent with the idea that proacrosin/acrosin remains associated to the acrosome reacted spermatozoa for long periods of time, and that proacrosin/acrosin associated to perivitelline spermatozoa could be responsible for the second penetration of fresh rabbit eggs by perivitelline spermatozoa.

Functional properties of caltrin proteins from seminal vesicle of the guinea pig

Caltrin proteins from seminal vesicle content of the guinea pig bind with great specificity to different regions of the spermatozoa. Indirect immunofluorescence studies with polyclonal antibodies showed that caltrin I binds to the head, on the acrosomal cup, while caltrin II binds on the principal tail and the neck. No fluorescence was detected either in the midpiece or in the post-acrosomal area of the head when sperm were exposed to either of the caltrins. Calcium-induced hyaluronidase release, which occurs during the acrosomal reaction, was dramatically inhibited by caltrin I (approximately 85% inhibition). Caltrin II was less effective in preventing the enzyme release (approximately 50% inhibition). Chemical modification of the structure modified the biological activity of the two caltrins. Reduction and carboxymethylation of the cysteine residues diminished the inhibitory activity on 45Ca2+ uptake and reduced the ability of the proteins to react with their antibodies. Removal of the carbohydrate portion by chemical deglycosylation transformed the inhibitor proteins into enhancers of calcium uptake into the spermatozoa. Caltrin proteins from the guinea pig appear to play the same physiological role as bovine caltrin, regulating specifically calcium transport across the spermatozoal membranes related with the acrosome reaction and hyperactivation process. The dual behavior of caltrins to inhibit or enhance Ca2+ uptake enables them to fulfill this function. Nevertheless, molecular mechanisms different from those described for bovine caltrin seem to be involved in the control of the functional activity of the guinea pig caltrins.

Localization of actin, alpha-actinin, and tropomyosin in bovine spermatozoa and epididymal epithelium.

Actin, alpha-actinin, and tropomyosin were localized in the testicular, epididymal, and ejaculated spermatozoa and in the epithelium of the bovine epididymis by means of specific antibodies using an indirect immunofluorescence technique. Immunocytochemical results were confirmed by the western blot analysis. Independent of the method of fixation, washing, or sonication, actin, alpha-actinin, and tropomyosin were all consistently localized in the neck of the spermatozoa. Actin and tropomyosin present in the postacrosomal area could be removed by sonication, whereas alpha-actinin in the basal plate appeared to be resistant to the treatment. In the unwashed spermatozoa alpha-actinin-specific immunofluorescence was seen over the acrosomal area, whereas in the washed sperm it appeared as a narrow cap at the margin of the head. In the latter location, its distribution was similar to that of tropomyosin. In the majority of preparations, tropomyosin could be localized in the principal piece of the tail. Even though some actin-specific immunofluorescence could be identified in the principal piece of the tail of the testicular and epididymal spermatozoa, a strong immunoreaction appeared only in the ejaculated spermatozoa. In the principal cells of the epididymal epithelium, specific fluorescence for actin, alpha-actinin, and tropomyosin occurred in the apical junctional complex. Basal bodies of the solitary cilia of the epididymal epithelium were labelled with antitropomyosin and anti-alpha-actinin antibodies. Besides offering new information about the cytoskeletal composition of the mammalian sperm, the present results support the hypothesized homology between the connecting piece of the sperm neck and the basal body of the cilia.

Species-specific localization of actin in mammalian spermatozoa: fact or artifact?

Actin has been characterized and localized in sperm cells of many mammals. Nevertheless, the reported localizations obtained by different methods and/or antibodies varied from species to species and even for the same species. To clarify the question, sperm actin distribution was reinvestigated under uniform technical conditions. Immunogold post-embedding procedures were performed using a polyclonal and two monoclonal antibodies of known specificity to localize actin in spermatids and spermatozoa of rabbit, mouse, rat, monkey, and human. In these species, actin (F-actin) was detected with the three antibodies between the nucleus and the acrosome of round and elongating spermatids. Species-specific changes occurred in maturing spermatids. In the rabbit, actin labeling decreased and disappeared from the tip to the base of the subacrosomal layer. In testicular and epididymal spermatozoa actin was detected only with a monoclonal antibody (Amersham) successively in the neck, postacrosomal area, and subacrosomal bulges. In mouse late spermatids a transitory labeling of the neck was detected only with the polyclonal antiactin. In testicular and epididymal spermatozoa an actin labeling was observed in the principal piece of the tail. In rat, monkey, and human sperm cells actin remained undetected. These results suggest that there is a redistribution of actin in late spermatids and spermatozoa which is a species-specific process but not an artifact of methodological origin. Thus, a function for actin in sperm, if any, remains to be demonstrated.

Viability and acrosome staining of bull, boar and rabbit spermatozoa.

A practical and reliable staining procedure was developed to distinguish the viability and acrosomal status of bull, boar and rabbit spermatozoa. The first stain with trypan blue or Congo red is rapid and avoids artifacts. This stain is precipitated by neutral red during the 2 min required for fixation. The precipitate gives a high contrast black color, resistant to the subsequent rinsing and persists during the time required for staining the acrosome with Giemsa. Ten classes of spermatozoa are distinguished (live or dead with intact acrosomes, loose acrosomes, damaged acrosomes, no acrosome, or with no acrosome and no postacrosomal ring). The intact acrosomes are purple, the loose acrosomes are dark lavender and the damaged acrosomes are pale lavender. The anterior part of the head of live spermatozoa with no acrosome is white or light pink and the same area of dead spermatozoa is white or pale gray. The postacrosomal ring is red. The postacrosomal area of the head of live spermatozoa is white or light pink and the same part of dead spermatozoa is black, dark violet or gray. The procedure did not give satisfactory results for stallion spermatozoa.

Surface sugar binding components of bovine spermatozoa as evidence by fluorescent neoglycoproteins.

In the present study, the topographical distribution of carbohydrate binding sites on the plasma membrane of bovine epididymal spermatozoa was investigated using 15 different fluorescent neoglycoproteins and asialoglycoproteins. With mannose-bovine serum albumin (BSA)-fluoresceinthiocarbamyl (FTC), mannose-6-phosphate-BSA-FTC, lactose-BSA-FTC, maltose-BSA-FTC, asialolactoferrin-FTC and asialotransferrin-FTC a marked fluorescence was observed in the postacrosomal area. These results further substantiate the concept that carbohydrate binding sites of the spermatozoan plasma membrane and corresponding carbohydrates of the zona pellucida play a significant role in gamete interactions.