Organization and modifications of sperm acrosomal molecules during spermatogenesis and epididymal maturation

The mammalian acrosome is a highly specialized organelle overlying the anterior part of the sperm nucleus and contains a variety of proteins, including hydrolytic enzymes and matrix molecules. Functionally, the anterior acrosome is involved in the acrosome reaction or sperm-zona pellucida interaction, while the equatorial segment (posterior acrosome) is involved in sperm-egg fusion. The acrosome is formed during spermiogenesis, during which associated molecules are transported from the Golgi apparatus and organized. Many of the molecules thus arranged gradually become compartmentalized during sperm passage through the epididymis. Some of them are further modified during the fertilization process. The findings indicate that acrosomal molecules are not only restricted to a specific region (domain) of the acrosome but also undergo ongoing relocation in a stage-specific manner during sperm maturation in the testis and epididymis. Such maturation-associated modifications are considered essential for sperm molecules to reach the correct or final site before fertilization. This review focuses on the organization and modifications of the acrosomal molecules as well as their compartmentalization within the acrosome.     

Biology of spermatozoa maturation: an overview with an introduction to this issue

Mammalian spermatozoa undergo morphological, biochemical, and physiological modifications initially in the testis (testicular maturation) and later in the epididymis (epididymal maturation). These maturational changes are commensurate with the functional events that occur in developing germ cells and maturing spermatozoa. This special issue reviews the recent, relevant topics dealing with spermatozoa maturation and focuses on the events that occur in internal components such as the nucleus, the acrosome, the perinuclear theca, the fibrous sheath, and the cytoplasmic droplet as well as the plasma membrane. These structures/elements and the constituent proteins of which they are comprised undergo a variety of sequential modifications starting from their origination in developing germ cells up to epididymal maturation. Several steps of the maturation processes on the sperm plasma membrane are mediated by external enzymes and secretions derived from the epithelium lining of the genital tract. Degradation of some of the constituent proteins and the elimination of defective spermatozoa are controlled by the degradation/recycling system, the ubiquitin system. These maturational modifications are necessary for spermatozoa to become fertilization-competent cells and to be stored safely in the male.     

Perinuclear theca during spermatozoa maturation leading to fertilization

Mammalian spermatozoa acquire the capacity to fertilize the ovum and display motility during their passage through the epididymis. At the same time, they undergo changes in metabolic patterns, enzymatic activities, ability to bind to zona pellucida surface, and electrophoretic properties and, furthermore, stabilization of some sperm structures by the establishment of disulphide linkages takes place in several sperm structures. The cytoplasmic perinuclear theca (PT) is a unique extranuclear cytoskeletal element that surrounds the nucleus, which is proposed to be a structural scaffold to the sperm nucleus. The purpose of this review is to describe PT changes related to epididymal sperm maturation. We will focus mainly on the protein components of the PT of eutherian mammalian spermatozoa and on quantitative protein changes during sperm maturation. The protein constituents of the PT have not been completely defined and most of them are different from the cytoskeletal proteins of somatic cells. However, they are proteins with cytoskeletal features. The morphologic changes reported for PT and the proposed functions of PT are discussed.     

Localizations of intracellular calcium and Ca2+-ATPase in hamster spermatogenic cells and spermatozoa

Calcium plays a predominant role regulating many functional processes of spermatogenesis and fertilization. The purpose of the present study is to define the exact location of calcium as well as examine the role it plays during spermatogenesis and sperm capacitation. Testes and epididymides were obtained from adult healthy male hamsters. Spermatozoa were incubated with modified Tyrode's medium up to 4 h at 37 degrees Celsius for sperm capacitation in vitro. Samples of the testes and sperm cells were analyzed by cytochemical techniques to determine the location of calcium and Ca(2+)-ATPase and the percentage of acrosome reactions under light and electron microscopy. The data showed that (1) Sertoli cells exhibited numerous calcium precipitates as large, round, electron-dense bodies distributed throughout the cytoplasm and the mitochondrial matrix. Fine calcium precipitates existed in fewer numbers in the intracellular storage sites of spermatogonia and primary spermatocytes, in sharp distinction to secondary spermatocyte and spermatids, which showed an abundance of large and round calcium precipitates, especially in the mitochondrial matrix of spermatids. More calcium deposits were distributed in the plasma membrane (PM), acrosome membrane, and matrices of the acrosome and mitochondria following capacitation; (2) Ca(2+)-ATPase was found in the endoplasmic reticulum system and PM of noncapacitated spermatozoa as well as Sertoli cells. Capacitated spermatozoa showed a weak signal. These results suggest that the presence of calcium in spermatogenic cells might play a role in cell growth and differentiation during spermatogenesis. The Ca(2+)-ATPase function may be inhibited during capacitation, leading to an increase in acrosomal calcium level and triggering of acrosomal exocytosis.     

Glycan modifying enzymes in luminal fluid of rat epididymis: are they involved in altering sperm surface glycoproteins during maturation?

Testicular spermatozoa undergo morphological and biochemical alterations, collectively termed epididymal maturation, in the intraluminal environment of epididymis. As a result of these modifications, the spermatozoon becomes a motile and functionally competent cell capable of undergoing capacitation and binding to the zona pellucida, the extracellular coat that surrounds the mammalian oocyte. Although details of all the changes are not fully known, several studies provide evidence suggesting that sperm plasma membrane undergoes extensive biochemical changes, including organization and modification of surface glycoproteins as spermatozoa transit from the proximal to the distal epididymis. In this article, I have attempted to summarize results with two sets of glycoprotein (glycan)-modifying enzymes, namely, glycohydrolases (hydrolytic enzymes) and glycosyltransferases (synthetic enzymes) present in the epididymal luminal fluid (LF). The in vitro experimental approaches described in this report demonstrate that: 1) a PNA-positive glycoprotein(s) (containing O-linked glycan) of 135-150 kDa subunit molecular mass which is present on the surface of caput (but not the cauda) spermatozoa can be degalactosylated by the enzymatic digestion with LF beta-D-galactosidase; and 2) an N-linked glycan chain(s) which is present on a sperm surface glycoprotein (apparent subunit molecular mass of 86 kDa) can be fucosylated in vitro when distal caput sperm (or sperm plasma membrane-rich fractions) are incubated in the presence of a nucleotide sugar (GDP[(14)C]fucose). Combined, these results strongly suggest a role for the glycan-modifying enzymes in degalactosylation and fucosylation of sperm surface glycoproteins during epididymal transit.     

Comparative fine structure of the epididymal spermatozoa from three Korean shrews with considerations on their phylogenetic relationships.

This study examined the fine structures of epididymal spermatozoa on the lesser white-toothed shrew (Crocidura suaveolens), the Japanese white-toothed shrew (C. dsinezumi) and the big white-toothed shrew (C. lasiura) belonging to the subfamily Crocidurinae living in Korea. In the spermatozoa of C. suaveolens, the head has a large acrosome, a smooth inner acrosomal membrane and a wavy, finger-like, electron-dense apical body. The neck has a solid proximal centriole that is filled with electron-dense material. These results showed the spermatozoa of C. suaveolens possess the characteristics of both Crocidurinae and Soricinae. In C. dsinezumi and C. lasiura, the head has a large acrosome, a serrated inner acrosomal membrane and a common apical body. The neck has a fistulous proximal centriole with slightly dense electron granules. These results showed the typical characteristics of Crocidurinae. Although C. suaveolens belongs to the subfamily Crocidurinae, the spermatozoan morphology is different from C. dsinezumi and C. lasiurai because it has conserved characteristics of the subfamily Soricinae.     

An ultrastructural study of spermiogenesis in two species of Sitophilus (Coleoptera: Curculionidae).

The spermiogenesis of Sitophilus zeamais and Sitophilus oryzae, the maize and the rice weevil, respectively, was studied by light microscopy and scanning and transmission electron microscopy. Sitophilus spp. is the most widespread and destructive primary pest of stored cereals in the world. The spermiogenesis occurs within cysts. There are approximately 256 germ line cells per cyst. Inside each cysts, all the spermatids are in the same stage of maturation. The ultrastructure of the spermatozoa of S. zeamais and S. oryzae is similar to that described for other beetles. The head is formed by a three-layered acrosome with the perforatorium, the acrosomal vesicle, the extra-acrosomal layer and the nucleus. The flagellum has the typical axoneme formed by a 9+9+2 microtubules arrangement, two mitochondrial derivatives and two accessory bodies. The typical pattern for Curculionidae spermatozoa described here may provide useful information for future phylogenetic analysis of the superfamily Curculionoidea.     

Development of a protocol for multiple staining with fluorochromes to assess the functional status of boar spermatozoa

The aim of this study was to design a simple and reliable method for the simultaneous evaluation of the nucleus, the acrosome, and the mitochondrial sheath of boar spermatozoa. Sperm samples coming from healthy and sexually mature Pietrain boars were incubated with two nuclear fluorochromes--bis-benzamide specific for viable cells, and propidium iodide specific for nonviable cells--the fluorochrome Mitotracker Green FM specific for functional mitochondria, and the lectin Trypsin inhibitor from Soybean (SBTI) conjugated with the fluorochrome Alexa Fluor 488 specific for proacrosin. The results obtained from assessing the functional status of the spermatozoa using fluorochromes were compared with the conventional sperm parameters of sperm vitality using the eosin exclusion test (EE test), and sperm motility and morphology using the computer-assisted semen analyzer SCA 2002Producció. Applying the multiple staining test, it was found that the frequency of viable spermatozoa with intact acrosome and intact mitochondria was not different from the frequency of viable spermatozoa obtained with the EE test, and also correlated positively with the frequency of motile spermatozoa and the frequency of mature spermatozoa. Therefore, this technique is useful to characterize the status of boar spermatozoa by assessing the nuclear, acrosomal, and mitochondrial integrity. Moreover, it provides reliable diagnostic information about the fertility potential of boars.     

Seasonal variations in the heterologous binding of viscacha spermatozoa. A scanning electron microscope study.

Seasonal changes in the reproductive activity of the adult male viscacha (Lagostomus maximus maximus) were investigated during the annual reproductive cycle. Assays of heterologous in vitro binding between compatible gametes were used to evaluate the ability of viscacha spermatozoa to achieve primary binding during its annual reproductive cycle. Sperm were collected by mincing cauda epididymis in HECM-3 medium and the sperm concentration and motility were evaluated. Cumulus-free and zona-free oocytes obtained from superovulated hamsters were inseminated in vitro with capacitated sperm suspensions, incubated at 37 degrees C, 5% CO2 for 3 h, and then processed for studies by scanning electronic microscopy. Statistical analysis was used to compare the quantitative differences. The number of spermatozoa significantly decreases during the regression period, while sperm motility was progressive speed in both periods. During the active period elevated sperm binding to cumulus-free and zona-free oocytes was observed, while the binding during the regression period decreased drastically. In both periods, oocyte microvilli covered sperm heads and tails. These results suggest that the ability of viscacha spermatozoa to participate in gamete recognition is profoundly affected. This would likely be related to different functional stages of the spermatozoa and their epididymal microenvironment during the annual reproductive cycle of viscacha.     

Cytoskeletal elements in mammalian spermiogenesis and spermatozoa.

Identification of the cytoskeletal elements and their role in the formation as well as the maintenance of head membrane compartmentalization is a much debated issue in mammalian spermatozoa. Data which have emerged during the last ten years are summarized. Those which have converged in a common opinion, such as the distribution of actin in mammalian spermiogenesis, are distinguished from those which have to be confirmed, such as the role of actin related proteins and actin in mature spermatozoa.     

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.     

Region‐specific localization of NOS isoforms and NADPH‐diaphorase activity in the intratesticular and excurrent duct systems of adult domestic cats (Felis catus)

Nitric oxide (NO) is produced by nitric oxide synthases (NOSs) and plays an important role in all levels of reproduction from the brain to the reproductive organs. Recently, it has been discovered that all germ cells and Leydig cells in the cat testis exhibit stage‐dependent nuclear and cytoplasmic endothelial (eNOS) and inducible (iNOS)‐NOS immunoreactivity and cytoplasmic nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐d) reactivity. As a continuation of this finding, in this study, cellular localization of NADPH‐d and immunolocalization and expression of all three NOS isoforms were investigated in the intratesticular (tubuli recti and rete testis), and excurrent ducts (efferent ductules, epididymal duct and vas deferens) of adult cats using histochemistry, immunohistochemistry and western blotting. NADPH‐d activity was found in the midpiece of the spermatozoa tail and epithelial cells of all of ducts, except for nonciliated cells of the efferent ductules. Even though the immunoblotting results revealed similar levels of nNOS, eNOS and iNOS in the caput, corpus and cauda segments of epididymis and the vas deferens, immunostainings showed cell‐specific localization in the efferent ductules and region‐ and cell‐specific localization in the epididymal duct. All of three NOS isoforms were immunolocalized to the nuclear membrane and cytoplasm of the epithelial cells in all ducts, but were found in the tail and the cytoplasmic droplets of spermatozoa. These data suggest that NO/NOS activity might be of importance not only for the functions of the intratesticular and excurrent ducts but also for sperm maturation. Microsc. Res. Tech. 79:192–208, 2016. © 2016 Wiley Periodicals, Inc.     

Zona Pellucida sperm-binding protein 3 receptor distribution during Gopc−/− globozoospermic spermatogenesis

Globozoospermia is a type of teratozoospermia characterized by round morphology of the sperm head. Gopc^−/− infertile globozoospermic murine model has failures during spermiogenesis, such as the incorrect biogenesis of the acrosome, disorganized acroplaxome and manchette, round nuclei and spiral flagella. In this study, Western blot, RT-PCR, immunohistochemistry and immunogold were done for the localization of the acrosome protein Zona Pellucida sperm-binding protein 3 receptor (ZP3R), also called sp56, in wild type and Gopc^−/− mice testis. The ZP3R protein was located in the acrosome and pseudo-acrosome vesicles of wild type and Gopc^−/− mice, respectively. Also, it is distributed through the cytoplasm of the haploid spermatids only. The incorrect spermiogenesis of Gopc^−/− mice causes a deregulation in the expression of ZP3R in the globozoospermic spermatids. Our results suggest that although the lack of GOPC causes a failure during the transport of the pre-acrosomal vesicles, the acrosome protein ZP3R is localized in the acrosome and is distributed through the cytoplasm only during spermiogenesis. Furthermore, the failure in spermiogenesis does not impair the synthesis of ZP3R and its localization in the pre-acrosomal vesicles.     

Ethanol exposure during peripubertal period increases the mast cell number and impairs meiotic and spermatic parameters in adult male rats

Puberty is characterized by psychosomatic alterations, whereas chronic ethanol consumption is associated with morphophysiological changes in the male reproductive system. The purpose of this study was to show the toxic effects on testis and epididymal morphophysiology after ethanol administration during peripuberty. To this end, male Wistar rats were divided into two groups: ethanol (E) group: received a 2 g dose of ethanol/kg in 25% (v/v); and control (C) group: received the same volume of filtered water; both were treated by gavage for 54 days. On the 55th day of the experiment, epididymis, and testis were collected for sperm count, histopathology, mast cell count, and morphometry. The vas deferens was collected for sperm motility analysis. The femur and testicle were used for cytogenetic analysis. Ethanol exposure caused reduction in daily sperm production (DSP) and in sperm motility, multinucleated cells or those having no chromosomal content, and late chromosome migrations. No changes were observed in the number of chromosomes in the mitotic analysis. However, some alterations could be seen in meiocytes at different stages of cell division. Stereological analysis of the epididymis indicated reorganization of its component in the 2A and 5A/B regions. The epididymal cauda had greater recruitment, and both degranulated and full mast cells showed an increase in the initial segment, in the ethanol group. In conclusion, ethanol administration during the pubertal phase affects epididymis and testis in adult rats, as indicated mainly by our new findings related to mast cell number and meiotic impact. Microsc. Res. Tech. 79:541–549, 2016. © 2016 Wiley Periodicals, Inc.     

Immunogold electron microscopic localization of antigenic sites in the outer dense fiber region of rat sperm tail obtained by using antisera to two Sertoli cell secretory proteins

Polyclonal antisera raised against polypeptide components of two rat Sertoli cell secretory proteins, designated protein S70 and S45–S35 heterodimeric protein on the basis of cell origin and estimated molecular weight, were used to identify antigenic sites in (1) rat testis, () cultured Sertoli cells, (3) developing spermatids (collected from spermatogenic stage-specific seminiferous tubular segments), and (4) epididymal sperm. Indirect immunofluorescence, immunoper-oxidase, and immunogold electron microscopy (single and double labeling) were used. Immunocytochemical techniques have detected antigenic sites in (1) the cytoplasm of Sertoli cells in the intact seminiferous tubule and in culture in the form of a punctuate, granular-like pattern, and (2) the acrosome (but not the Golgi region) and tail of developing spermatids and sperm. In developing spermatids, the principal piece of the tail displays a characteristic apical-to-distal immunoreactive banding pattern that correlates both temporally and spatially with the reported multistep assembly of outer dense fibers along the axoneme. The immunoreactivity of the acrosome, connecting piece, and outer dense fibers of the sperm tail was confirmed by immunogold electron microscopy. A precise identification of the component(s) of the outer dense fiber region responsible for the antigenic homology with Sertoli cell secretory proteins is under investigation.     

Protein detection in spermatids and spermatozoa of the butterfly Euptoieta hegesia (Lepidoptera).

This study was undertaken to detect protein components in both sperm types of the butterfly Euptoieta hegesia. These spermatozoa possess complex extracellular structures for which the composition and functional significance are still unclear. In the apyrene sperm head, the proteic cap presented an external ring and an internal dense content; basic proteins were detected only in external portions. In the tail, the paracrystalline core of mitochondrial derivatives and the axoneme are rich in proteins. The extratesticular spermatozoa are covered by a proteic coat, which presented two distinct layers. In eupyrene spermatozoa, acrosome and nucleus were negatively stained, probably because of their high compaction. In the tail, there is no paracrystalline core and the axoneme presented a very specific reaction for basic proteins. The lacinate and reticular appendages are composed of cylindrical sub-units and presented a light reaction to E-PTA and a strong reaction to tannic acid. A complex proteic coat also covers the extratesticular spermatozoa. We found similarities between both extratesticular coats, indicating a possible common origin. Both spermatozoon types are rich in proteins, especially the eupyrene appendages and the extratesticular coats. We believe that both coats are related to the sperm maturation and capacitation processes.     

Ultrastructural characteristics of spermatogenesis in Pallas's mastiff bat, Molossus molossus (Chiroptera: Molossidae)

Despite the large number of species, their wide distribution, and unique reproductive characteristics, Neotropical bats have been poorly studied, and important aspects of the reproduction of these animals have not been elucidated. We made an ultrastructural analysis of spermatogenesis in Molossus molossus (Molossidae). The process of spermatogonial differentiation is similar to that found in other bats and is also relatively similar to that of Primates, with three main spermatogonia types: A(d), A(p), and B. Meiotic divisions proceed similarly to those of most mammals, and spermiogenesis is clearly divided into 12 steps, in the middle of the range known for bats (9-16 steps). Formation of the acrosome is similar to that known from other mammals; however, the ultrastructure of spermatozoa was found to have unique characteristics, including many wavy acrosomal projections on its surface, which seems to be specific for the family Molossidae. Comparing the ultrastructure of the spermatozoon of M. molossus with other bats already study, we observed that three characters vary: morphology of the outer dense fibers, of the perforatorium, and of the spermatozoon head. The great similarity of morphological characters between M. molossus and Platyrrhinus lineatus suggests that M. molossus is more closely related to the Phyllostomidae than to the Rhinolophidae and the Vespertilionidae.     

Sperm ultrastructure of Mytella (Bivalvia) populations from distinct habitats along the northern coast of São Paulo State,Brazil

Ultrastructural analyses of bivalve spermatozoa are relevant in studies that aim to identify taxonomic traits for the purposes of discriminating species and conducting phylogenetic studies. In the present work, spermatozoa of mussel specimens of the genus Mytella, collected from two populations living in distinct habitats, were examined by electron microscopy. The objective was to identify sperm ultrastructural taxonomic traits that could be used to differentiate Mytella species. The specimens were from populations that live in intertidal zones on the southeast coast of Brazil, either buried in muddy-sand sediment or anchored to rocky substrates. The acrosomal vesicle was conical and long, the axial rod extended from the nucleus to the acrosome, the nucleus was an oblate spheroid with a condensed chromatin, the intermediate portion contained mitochondria encircling a pair of centrioles, and there was a single flagellum. The sperm was of a primitive type. The spermatozoon ultrastructure did not distinguish the specimens buried in muddy-sand sediment from those anchored to rocky substrates. The data suggest that the specimens analyzed, despite living in distinct habitats, belong to the same species, which conchological analyses identified as M. charruana. The presence of an axial rod in their sperm cells supports the inclusion of M. charruana in the subfamily Mytilinae.     

Ultrastructure of spermatozoa in two solitary bee species with an emphasis on synapomorphic traits shared in the family apidae

Morphology of spermatozoa in bees has provided promising results for phylogenetic analyses. In this work, the structure and ultrastructure of spermatozoa from Thygater (Thygater) analis and Melitoma segmentaria were characterized and the synapomorphies shared in the family Apidae are discussed. In these species, spermatozoa bundles which are undone in the seminal vesicle possess, on average, 50 cells. Spermatozoa consist of a head and a flagellar region. The head includes an acrosome containing the perforatorium, covered by the acrosomal vesicle and a nucleus. The flagellum is formed by two mitochondrial derivatives, which are asymmetric in diameter and length, with one centriolar adjunct, one axoneme (9 + 9 + 2), and two accessory bodies. In cross section the centriolar adjunct is asymmetric and the accessory bodies are triangular in shape. In the distal region of the flagellum, the derivative terminates before the axoneme and the small derivative terminates first. The axoneme is gradually disorganized and the accessories microtubules are the last to terminate. In these two species, spermatozoa share diverse synapomorphies with those of other bee species previously described in the literature, which allows for the establishment of a morphological pattern for spermatozoa of the family Apidae.