Structural modification of the hamster sperm acrosome during posttesticular development in the epididymis

The acrosome of the mature spermatozoon functions as a regulated secretory vesicle which performs several critical functions in mammalian fertilization. Acrosome assembly occurs throughout spermiogenesis and continues during posttesticular sperm maturation in the epididymis, resulting in a structurally polarized membrane-bounded organelle that contains an assortment of hydrolases and a stable infrastructure termed the acrosomal matrix. The role of stable acrosomal matrix assemblies in acrosomal biogenesis and function are poorly understood. This article presents ultrastructural, immunocytochemical, and biochemical data on the remodeling of the hamster acrosomal matrix during spermiogenesis and posttesticular sperm maturation in the epididymis. Specific posttranslational modifications of the major acrosomal matrix protein are evident in late, step 16, spermatids and matrix protein processing continues within specific acrosomal subdomains of caput epididymal spermatozoa. At the completion of sperm maturation, the acrosomal matrix consists of two structurally distinct domains which are adherent to the outer acrosomal membrane and exhibit a localized distribution pattern. Coincident with acrosomal matrix differentiation, a paracrystalline cytoskeletal complex is assembled onto the outer acrosomal membrane of epididymal spermatozoa. This cytoskeletal network appears to establish transmembrane structural interactions with the acrosomal matrix and may maintain attachment of the acrosomal cap to the sperm head during the early steps of the acrosome reaction.     

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.     

Ubiquitin-dependent proteolysis in mammalian spermatogenesis,fertilization, and sperm quality control: killing three birds with one stone

Ubiquitin and ubiquitin-like proteins control the degradation of substrates as diverse as cyclins, viral envelope proteins, plasma membrane receptors, and mRNAs. The ubiquitinated substrates are targeted towards the lysosomal or proteasomal degradation sites. The number and position of ubiquitin molecules bound to substrates' lysine residues and the number and position of ubiquitin molecules in polyubiquitin chains determine the astonishing substrate specificity of ubiquitin-mediated proteolysis. Ubiquitin is likely to be expressed in mammalian gametes and embryos at any given developmental step, but the information on ubiquitin dependence of gametogenesis and fertilization is sketchy. Ubiquitin ligases E1, E2, E3, and UBC4 are active in the testis. Ubiquitin and proteasomal subunits can be detected in the human sperm centrosome that undergoes dramatic reduction during spermatid elongation. Spermatid histones are ubiquitinated as they are being transiently replaced by transitional proteins and permanently by protamines. Ubiquitin tagging of the sperm mitochondrial membranes may serve as a death sentence for paternal mitochondria at fertilization, thus promoting the maternal inheritance of mitochondrial DNA (mtDNA) in mammals. The defective spermatozoa become surface-ubiquitinated during sperm descent down the epididymis. Finally, new evidence suggests the involvement of ubiquitin-proteasome pathway in the zona penetration by the acrosome-reacted spermatozoon. Such differential patterns of ubiquitination in the testis and epididymis, and inside the egg, may be necessary for reproductive success in humans and animals. Deciphering and eventually manipulating the ubiquitin-dependent proteolysis in the reproductive system could allow us to redirect the mode of mtDNA inheritance after cloning and ooplasmic transplantation, provide germ line therapy in some cases of male infertility, develop new contraceptives, manage polyspermia during in vitro fertilization, and establish objective markers for infertility diagnostics, semen evaluation, and prediction of future fertility.     

Polyspermy prevention in marine invertebrates

In marine invertebrates, as in most other organisms, normal development requires that only one sperm nucleus joins with the egg nucleus at fertilization. The principal mechanisms employed are (1) prevention of sperm-egg plasma membrane fusion and (2) modifications of the egg extracellular coat to prevent sperm binding and/or penetration. In a third strategy, fertilization is polyspermic, but only one sperm nucleus fuses with the egg nucleus. Other factors such as gamete density during spawning, chemotaxis, and localized sites for sperm entry may also affect the numbers of sperm reaching the egg.     

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.     

Ultrastructural analysis of sperm from the genus <i>Idarnes</i> (Hymenoptera: Chalcidoidea,Sycophaginae)

In this study, the sperm ultrastructure of three species of Idarnes genus was investigated using light and transmission electron microscopy. Spermatozoon morphology of the three species was similar to that of most Chalcidoidea, with helicoidally twisted nucleus and flagellum. The head region consists of an acrosome and a nucleus; the nucleus-flagellum transition region characterized by the presence of mitochondrial derivatives and the centriolar adjunct; a flagellum region, which includes the axoneme with microtubular arrangement 9 + 9 + 2 and two mitochondrial derivatives. However, the sperm of these three species exhibit features that discriminate one species from each other: (1) only one species, Idarnes sp. 2 (carme group) exhibited an extracellular sheath surrounding the anterior portion of the nucleus, which extends to the anterior region of the flagellum, but it did not present filaments; (2) the acrosome in the three species was quite different, Idarnes sp. 1 and Idarnes sp. 2 (carme group) has two compartments (acrosomal and subacrosomal vesicles) while Idarnes sp. 3 (flavicollis group) has a third compartment (perforatorium); (3) the centriolar adjunct elongated and its location among the mitochondrial derivatives is similar for the three species analyzed; (4) mitochondrial derivatives differ between the species, with triangular (Idarnes sp. 1 and sp. 3) and elongated or flat shaped (Idarnes sp. 2) appearance. These data shows that sperm structure may differ within the same genus and confirms the potential of these cells in phylogenetic and taxonomic analyses in the Chalcidoidea superfamily, as well as in Hymenoptera in general.     

Expression of the male reproduction‐related gene in spermatic ducts of the blue swimming crab,Portunus pelagicus,and transfer of modified protein to the sperm acrosome

Expression of a sex‐specific gene in Macrobrachium rosenbergii (Mr‐Mrr), encoding a male reproduction‐related (Mrr) protein, has been identified in the spermatic ducts (SDs) and postulated to be involved in sperm maturation processes. M. rosenbergii is the only decapod that the expression and fate of the Mrr protein has been studied. To determine that this protein was conserved in decapods, we firstly used cloning techniques to identify the Mrr gene in two crabs, Portunus pelagicus (Pp‐Mrr) and Scylla serrata (Ss‐Mrr). We then investigated expression of Pp‐Mrr by in situ hybridization, and immunolocalization, as well as phosphorylation and glycosylation modifications, and the fate of the protein in the male reproductive tract. Pp‐Mrr was shown to have 632 nucleotides, and a deduced protein of 110 amino acids, with an unmodified molecular weight of 11.79 kDa and a mature protein with molecular weight of 9.16 kDa. In situ hybridization showed that Pp‐Mrr is expressed in the epithelium of the proximal, middle, distal SDs, and ejaculatory ducts. In Western blotting, proteins of 10.9 and 17.2 kDa from SDs were all positive using anti‐Mrr, antiphosphoserine/threonine, and antiphosphotyrosine. PAS staining showed they were also glycosylated. Immunolocalization studies showed Pp‐Mrr in the SD epithelium, lumen, and on the acrosomes of spermatozoa. Immunofluorescence staining indicated the acrosome of spermatozoa contained the Mrr protein, which is phosphorylated with serine/threonine and tyrosine, and also glycosylated. The Mrr is likely to be involved in acrosomal activation during fertilization of eggs. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

Electron microscopic observation of the sagittal structure of Drosophila mature sperm

Observation of sperm development and determination of their morphological characteristics are very important to the understanding of phylogenetic relationships and the study of sperm function during fertilization. Although ultrastructural studies of sperm development in the testes of the fruit fly Drosophila have been performed, there are few reports describing electron microscopic morphology of mature sperm, that is, those released from the testes to the seminal vesicles. Here, we present the first report of the sagittal organization of Drosophila sperm head and neck regions by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The head and tail structures of a mature sperm, for example, the acrosome, nucleus, and flagellum, were easy to distinguish by the morphological characteristics of the sperm surface by SEM. The morphological relationships between the surface and internal structures of mature sperm were confirmed by observing longitudinal sections with TEM. Our approach overcame the technical difficulties involved in sample preparation for electron microscopic observation of the Drosophila mature sperm head, and therefore, this study serves as an important foundation for future genetic dissection of sperm ultrastructure and function in male sterile mutants. Microsc. Res. Tech. 77:661–666, 2014. © 2014 Wiley Periodicals, Inc.     

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.     

Morphology of mammalian sperm membranes during differentiation,maturation, and capacitation

The mammalian spermatozoon is a highly polarized cell whose surface membrane can be divided into five functionally, structurally, and biochemically distinct domains. These domains are formed during spermatogenesis, continue to be modified during passage through the epididymis, and are further refined in the female reproductive tract. The integrity of these domains appears to be necessary for the sperm to perform its function—fusion with the egg and subsequent fertilization. The domains can be identified morphologically by their surface contours and texture, the content, distribution, and organization of intramembranous particles after freeze-fracture, and by the density of surface and cytoplasmic electron-dense coatings in thin sections. By using a variety of labels that stain carbohydrates (lectins), lipids (filipin and polymyxin B), and monoclonal antibodies to specific membrane constituents, the biochemical composition of these contiguous membrane regions has also been partly elucidated. We review here what is known about the structure, composition, and behavior of each membrane domain in the mature sperm and include some information regarding domain formation during spermatogenesis. The sperm is an excellent model system to study the creation and maintenance of cell polarity, granule exocytosis, and fertilization. Hopefully this review will provide impetus for future studies aimed more directly at addressing the relationship of its morphology to its functions.     

Morphological aspects of testes and sperm ultrastructure in the "symphyta" Digelasinus diversipes kirby 1882 (hymenoptera: Argidae: Dielocerinae)

In Digelasinus diversipes, spermatozoa are maintained in bundles, with 74 spermatozoa on average, in the seminal vesicle. These spermatozoa are very short (20 μm) and consist of a head and flagellum. The head includes an acrosome (perforatorium covered by the acrosomal vesicle) and a nucleus. A regular electron-lucent region separates the acrosomal vesicle from the perforatorium, which is inserted parallel to the anterior ending of the nucleus. The small flagellum is composed of two symmetrical mitochondrial derivatives, a centriolar adjunct, an axoneme (9 + 9 + 2), and two accessory bodies. The centriolar adjunct begins above the posterior end of the nucleus and ends covering the anterior tip of two mitochondrial derivatives. In the terminal region of the axoneme, the central microtubules terminate first. The presence of a subacrosomal space, a short mitochondrial derivative diameter, and a short spermatodesm is the ultrastructure characteristics of spermatozoa shared by all "symphyta" species. Differences in the insertion of the perforatorium into the nucleus and the position of the centriolar adjunct distinguish Dielocerinae and the Arginae studied previously. The number of spermatozoa per cyst is variable. Furthermore, additional characteristics that had not been described for "symphyta" were also found, such as the number of follicles per testis.     

Structural and ultrastructural characterization of zebu (Bos indicus) spermatozoa.

The ultrastructure of normal, ejaculated spermatozoa of Bos indicus was studied by means of electron microscopy, being evaluated in two principal parts, the head and the tail. The head is flat, oval or paddle-shaped with a square base, which provides a concave recess for the insertion of the tail. The acrosome tightly covers the anterior two thirds of the nucleus. A distinct unilateral acrosomal bulge was observed along the apical edge of the head. The equatorial region demarcates the acrosome from the post-equatorial region that covers the caudal one third of the nucleus. The classical 9+9+2 fiber pattern which composes the axoneme was observed along three segments of the tail, namely middle, principal and terminal pieces. The axoneme is anteriorly bound by the mitochondrial helix (middle piece) and posteriorly by the fibrous helix (principal piece), except at the terminal piece. The border between the middle piece and principal piece was well defined due to the termination of the thick mitochondrial helix and the presence of the annulus. Some of the spermatozoa presented cytoplasmatic droplets, which appeared as stalk-like appendages.     

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.     

Glycoconjugates of the urodele amphibian testis shown by lectin cytochemical methods

Lectin histochemistry is a useful method that allows the in situ identification of the terminal sugar moieties of the carbohydrates that form the glycoconjugates. Moreover, when it is combined with chemical or enzymatic deglycosylation pretreatments, lectin histochemistry can be employed to determine if carbohydrates are linked to the protein core by means of an N- or O-glycosidic linkage or, indeed, to partially sequence the sugar chains. One of the most interesting model organs for the study of spermatogenesis is the amphibian urodele testis. However, this organ has not been very widely investigated with lectin histochemical research. In the last few years, we have carried out a research project to identify and locate glycoconjugates in the testis of the urodele Pleurodeles waltl, the Spanish newt, as a first approach to identify possible carbohydrates with key roles in spermatogenesis. Our findings reveal some glycan chains located in a fusome-like structure in early (diploid) germ cells, oligosaccharides with terminal GalNAc in the acrosome, the occurrence of glycan modifications in the acrosomal contents during spermiogenesis, and changes in glycan composition of follicle and interstitial cells during the spermatogenetic cycle. Furthermore, the similar labeling pattern of follicle and duct cells supports the hypothesis for a common origin of both cell types.     

Sperm ultrastructure of Panorpodes kuandianensis (Mecoptera: Panorpodidae)

The sperm ultrastructure of the short‐faced scorpionfly Panorpodes kuandianensis Zhong et al. was investigated using transmission electron microscopy. The spermatozoon is composed of a short head containing a bilayered acrosome and an elongate nucleus, a neck transition region, and a long flagellum. The acrosome consists of an acrosomal vesicle and a central perforatorium. The nucleus has two deep, U‐shaped lateral grooves and its chromatin contains a series of parallel, regularly arrayed nuclear fibers. The barrel‐shaped centriolar adjunct occupies the most volume of the neck region. The flagellum is helical for its whole length and is formed by an axoneme, two mitochondrial derivatives, a pair of accessory bodies, and peculiar accessory structures. The axoneme has nine accessory microtubules at the anterior flagellum region, displaying a 9 + 9 + 2 microtubular pattern, but the accessory microtubules are short and disappear quickly. The two mitochondrial derivatives differ in length and diameter. In the more posterior region, the remaining anchor‐shaped mitochondrial derivative has a circular crystalline region, differing from other mecopteran species. The filiform materials are peculiar and lie beside the nucleus and in the flagellum region. Sperm ultrastructural comparison of P. kuandianensis with other families supports a close affinity of Panorpodidae with Panorpidae. In addition, the occurrence of the nine accessory microtubules in the sperm axoneme of Panorpodes indicates that the 9 + 9 + 2 axonemal pattern might be a symplesiomorphy of the Mecoptera. Microsc. Res. Tech. 77:394–400, 2014. © 2014 Wiley Periodicals, Inc.     

Interactions of sperm perinuclear theca with the oocyte: implications for oocyte activation,anti-polyspermy defense,and assisted reproduction

Perinuclear theca (PT) is the cytoskeletal coat of mammalian sperm nucleus that is removed from the sperm head at fertilization. PT harbors the sperm borne, oocyte-activating factor (SOAF), a yet-to-be-characterized substance responsible for triggering the signaling cascade of oocyte activation, thought to be dependent on intra-oocyte calcium release. The present article reviews the current knowledge on the biogenesis and molecular composition of sperm PT. Possible functions of sperm PT during natural and assisted fertilization, and in the initiation of embryonic development are discussed. Furthermore, evidence is provided that SOAF is transferred from the sperm PT to oocyte cytoplasm through the internalization and rapid solubilization of the post-acrosomal PT. It is shown that during natural fertilization the sperm PT dissolves in the oocyte cytoplasm concomitantly with sperm nuclear decondensation and the initiation of pronuclear development. SOAF activity is preserved in the differentially extracted sperm heads only if the integrity of PT is maintained. After intracytoplasmic sperm injection (ICSI), activation occurs only in those oocytes in which the injected spermatozoon displays complete or partial dissolution of PT. In the latter case, the residual PT of the sub-acrosomal and/or post-acrosomal sperm region may persist on the apical surface of the sperm nucleus/male pronucleus and may cause a delay or arrest of zygotic development. We propose that the sperm PT harbors SOAF in the post-acrosomal sheath, as this is the first part of the sperm cytosol to enter the oocyte cytoplasm and its disassembly appears sufficient to initiate the early events of oocyte activation. Dissolution of the sub-acrosomal part of the PT, on the other hand, appears necessary to insure complete DNA decondensation in the internalized sperm nucleus and initiate DNA synthesis of both pronuclei. The release of the SOAF from the sperm head into oocyte cytoplasm at fertilization ultimately leads to the activation of oocyte mechanism including the completion of the meiotic cell cycle, pronuclear development and anti-polyspermy defense.     

How does polyspermy happen in mammalian oocytes?

Polyspermy is one of the most commonly observed abnormal types of fertilization in mammalian oocytes. In vitro fertilization (IVF) provides approaches to study the mechanisms by which oocytes block polyspermic fertilization. Accumulated data indicate that oocyte, sperm and insemination conditions are all related to the occurrence of polyspermic fertilization. A high proportion of immature and aged oocytes showed polyspermy as compared with mature oocytes. Preincubation of oocytes and/or sperm with oviductal epithelial cells or collected oviductal fluid before IVF reduces polyspermic penetration. Recently, it was found that an abnormal zona pellucida is one of main causes of polyspermy in human eggs. A high proportion of polyspermy has resulted from the use of a high concentration of capacitated spermatozoa at the site of fertilization, irrespective of in the in vivo or in vitro environment. Oviductal secretions or oviductal epithelial cells themselves can regulate the number of spermatozoa reaching or binding to the zona pellucida thus reducing multiple sperm penetration. Suboptimal in vitro conditions, such as supplementations in IVF media, pH, and temperature during IVF, also induce polyspermic fertilization in some mammals. Species-specific differences are present regarding the relationship between insemination conditions and polyspermy.     

Proacrosin-acrosin activity in capacitated and acrosome reacted sperm from cryopreserved bovine semen.

Acrosin activity is associated with normal fertility in human and bovine spermatozoa. The aim of the study was to determine the variation of the enzyme activity in the proacrosin-acrosin system in capacitated and acrosome reacted cryopreserved bovine sperm. Enzyme activity was assessed spectrophotometrically using N-alpha-benzoyl-DL-arginine p-nitroanilide (BAPNA) as specific substrate for acrosin at pH 8. Capacitation with heparin and quercitin failed to induce conversion of proacrosin to acrosin. An increase in acrosin activity produced by the presence of progesterone, in a dose-dependent manner, was related with the induction of true acrosome reaction. The total level of acrosin activity registered showed that 96% of acrosin of capacitated sperm samples and control is present in the zymogen form. Moreover, progesterone is capable of duplicating the level of active enzyme, indicating that enzyme activity changes are related to acrosome reaction, suggesting that only a minor proportion of the total of proacrosin-acrosin system is required in the exocytotic process on cryopreserved bovine sperm.     

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.     

Fertilization-Induced changes in the vitelline envelope of echinoderm and amphibian eggs: Self-assembly of an extracellular matrix

The surface of the unfertilized sea urchin egg is covered by the vitelline layer (VL), a fibrous extracellular matrix that contains receptors for sperm. At fertilization, cortical granule exocytosis releases enzymes and structural proteins that cause the VL to elevate and become remodelled into the mechanically and chemically tough fertilization envelope. This envelope prevents further penetration of sperm and protects the embryo during early development. A thicker, more complex vitelline envelope surrounds the Xenopus laevis egg. This fibrous coat is also restructured at fertilization to produce an impenetrable barrier to sperm. The biochemical steps that occur during self-assembly of these fertilization envelopes are reviewed, and the ultrastructural changes that occur, as seen in platinum replicas of quick-frozen, deep-etched, and rotaryshadowed eggs, are illustrated.