Ultrastructural characteristics of the spermatogenesis during the four phases of the annual reproductive cycle of the black myotis bat,Myotis nigricans (Chiroptera: Vespertilionidae)

Myotis nigricans is an endemic species of vespertilionid bat, from the Neotropical region, that resembles temperate zone bats in their reproductive cycle; presenting an annual reproductive cycle with two periods of testicular regression, which are not linked to the apoptotic process and seems to be not directly linked to any seasonal abiotic variation. Thus, this study aimed to ultrastructurally evaluate their reproductive cycle. The process of testicular regression could be divided into four periods: active; regressing; regressed and recrudescence; with all presenting distinct characteristics. The active period was similar to that of other bats, presenting the complete occurrence of spermatogenesis, with three main types of spermatogonia (A_d, A_p, and B) and 12 steps in spermatid differentiation; however, it differed in having the outer dense fibers 1, 5, 6, and 9 larger than the others. These three types of spermatogonia undergo considerable morphologic changes from regressing to the regressed period, and in the recrudescence, they return to the basic morphology, which reactivates spermatogenesis. In conclusion, our study described the process of spermatogenesis, the ultrastructure of the spermatozoa and the distinct morphologic variations in the ultrastructure of the testicular cells of M. nigricans during the four different periods of its annual reproductive cycle. Microsc. Res. Tech., 76:1035–1049, 2013. © 2013 Wiley Periodicals, Inc.     

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.     

Cyclic nucleotide phosphodiesterase inhibition increases tyrosine phosphorylation and hyper motility in normal and pathological human spermatozoa.

Our objective was to determine the effect of phosphodiesterase (PDE) inhibition on: 1) tyrosine phosphorylation of human spermatozoa at the tail level; and 2) sperm motion parameters and hyperactivated motility. The study was conducted with normozoospermic and asthenozoospermic samples incubated under in vitro capacitating conditions. The main outcome measures were computer-assisted sperm motion analysis and fluorescent immunodetection of phosphotyrosine-containing proteins. Pentoxifylline (PTX) was used as PDE inhibitor because of its wide use in the clinic. PTX-treatment significantly increased sperm velocity, hyperactivated motility and tyrosine-phosphorylation, both in normo and asthenozoospermic samples. Tyrosine-phosphorylation of tail proteins was highly conspicuous in both types of samples, showing no differential pattern after PTX-treatment. Normozoospermic samples treated with pentoxifylline showed an increase in the number of spermatozoa displaying hyperactivated movement and tyrosine-phosphorylation at the tail level. Preliminary data on asthenozoospermic samples exhibiting altered motion characteristics and defective phosphorylation of sperm-tail proteins showed that both defects can be concomitantly overcome by pentoxifylline treatment. Tyrosine-phosphorylation of sperm-tail proteins is underlying the enhancement of hyperactivated motility resulting from PDE inhibition by pentoxifylline.     

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.     

In vitro fertilization and polyspermy in the pig: factors affecting fertilization rates and cytoskeletal reorganization of the oocyte

Polyspermy is a common phenomenon in the pig. Extensive information has become available from in vitro studies on not only the quality of oocytes but also the quality of spermatozoa. However, little information is available on the relative penetration rates of fresh and frozen spermatozoa from the same ejaculate from boars of different breeds. The present results, based on a total of 15 boars of three different breeds, revealed that the inter-breed variation in fertilization and polyspermic rates is larger than intra-breed variation. It was also shown that the incidence of polyspermy as well as penetration rate was greatly decreased by freezing and thawing, even if a higher number of sperm was coincubated with cumulus-free oocytes for a longer period compared to fresh sperm of the same ejaculate. This study focuses on the cytoskeletal organization of the oocyte with respect to the status of cumulus investment, and monospermic and polyspermic fertilization. The status of cumulus cells correlated with the density of transzonal cumulus-cell processes and with the maturation rate of oocytes and, to some degrees, the incidence of polyspermy. Polyspermic zygotes formed multiple microtubule domains in association with individual male pronuclei (PN), but in a high degree of polyspermy (more than trispermy), the pronuclear apposition did not proceed. The effect of multiple PN of paternal and maternal origin on the cytoskeletal reorganization is also discussed.     

The ovary of Lagostomus maximus (Mammalia, Rodentia): an analysis by confocal microscopy

Lagostomus maximus is a notable mammalian model for reproductive studies. Females have an extremely high ovulation rate, which is due to down-regulation of the follicular apoptosis pathway, which ensures a large pool of developing follicles. This large pool is supported by the convoluted anatomy of the mature ovary, whose germinal tissue is found in irregularly curved ridges throughout the cortex. Medullary tissue is restricted to a minimum. Lyso Tracker Red reconstruction under confocal laser scanning microscopy was used to recognize and measure all follicular stages from primordial to antral. Unlike most mammals in which early primordial follicles are just found in fetal life, the adult ovary shows regions packed with early primordial follicles. Follicle size ranged from 24 to 316 microm. We discuss the relationships of L. maximus follicles size with regard to other species of mammals and propose that the physiology of the adult viscacha ovary obeys to a neoteny process in the evolution of this species.     

Single-channel response of hamster oocytes to fertilization with homologous spermatozoa.

Electrophysiological events occur early after fertilization, along with changes in intracellular Ca2+ concentration. Passive electrical parameters were determined in golden hamster oocytes by whole cell patch-clamp method. In separate experiments the effect of 4-aminopyridine on resting oocytes was tested. The single-channel patch clamp configuration was employed to assess the electrical response to fertilization with homologous sperm. Structure of oocytes submitted to patch clamp was evaluated with scanning electron microscopy and found to be preserved. Oocyte diameter was 70.2 +/- 2.2 microm; their resting parameters were: membrane potential 23.8 +/- 0.8 mV; total membrane specific resistance 519.1 +/- 94.6 ohms.cm2, and specific capacity 0.99 +/- 0.03 microF.cm(-2). Total membrane current was decreased by 42 % by 4-aminopyridine. Control oocytes and oocytes exposed to sperm differed in their membrane currents in response to a voltage ramp clamping membrane potential from - 100 mV to + 100 mV. In both cases, currents were largest at the most negative potentials, but sperm-exposed oocytes had larger currents. Additionally, while in control oocytes the current was inward at negative potentials but outward at positive potentials, in the presence of spermatozoa oocytes was inward within the whole voltage range tested. This latter current may represent Ca2+ entry.     

Characterization of Albanian water frog,Pelophylax shqipericus,sperm traits and morphology,by using phase contrast microscopy

Mature spermatozoa traits and morphology of endangered Albanian water frog, Pelophylax shqipericus, have been characterized for the first time through phase contrast microscopy, as part of successful implementation of in vitro fertilization technique for this species. The basic morphology of P. shqipericus spermatozoa consists of an elongated, thick, smooth‐edged, and solid‐staining head, continuing with a thin and long tail which usually extends 2.48 times the head length. The acrosome was not clearly discernible so the measurements were done on the head as a whole, while the middle section was better visible. Average length of head, including the acrosome and midsection was estimated to be 11.78 μm ± 0.32, while the tail length resulted 29.24 ± 1.75 μm. The average thickness of the head was shown to be 3.45 μm. The total sperm length resulted to be 41.02 ± 1.83 μm. The average sperm concentration was estimated of 25.5 × 10⁶/ml. Sperm amount, survival rate and motility were also measured. The sperm survival rate was maximal immediately after preparation of the suspension and tended to decrease over time of storage, reaching 50% after 72 hr. Decreased sperm motility seemed to follow the same trend as sperm viability. Sperm traits resulted to be very similar both in size and in shape with those of “Lessonae” frog group, one of the lineages of Western Palearctic species complex, suggesting a strong phylogenetic relationship among these species.     

Quantification of human sperm morphology and motility by means of semi-automatic image analysis systems.

Sperm morphology and motility are determined using a semi-automatic image analysis system under opto-manual control. The system consists of a microscope equipped with a drawing-tube, a digitizer-tablet, and a cursor interfaced to a small microcomputer. A light-emitting diode is mounted on the cursor, visible as a bright red pilot-light in the microscopic field. Sperm head and midpiece as well as the pathway of motile spermatozoa are traced with the cursor's red pilot light. The microcomputer calculates area, circumference, and length simultaneously. Motility and sperm density are determined altogether using microchamber technique. In a selected group of male subjects with normal, doubtful, and pathological semen analyses biometrical analyses of sperm morphology and motility were performed. Sperm morphology is best described by the variation of the head size and by the average midpiece width. Motility is best described by % motile spermatozoa and mean velocity determined 1 hour after semen collection. Biometrical semen analyses are superior to subjective evaluations regarding degree of information, objectivity, and reproducibility.     

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.     

Incidence of sperm-tail tyrosine phosphorylation and hyperactivated motility in normozoospermic and asthenozoospermic human sperm samples

Our objective was to study the incidence of sperm-tail phosphotyrosine immunoreactivity in normozoospermic and asthenozoospermic human sperm samples, its association with sperm motion parameters, particularly hyperactivated motility, and its potential involvement in the pathogenesis of asthenozoospermia. The work was conducted as a prospective experimental study in the Sperm Biology and Andrology laboratories of the Jones Institute, a medical school-based fertility center. The study subjects were healthy fertile male donors (normozoospermic samples) and infertile patients (asthenozoospermic samples) attending the center. Recently ejaculated semen samples were washed twice to eliminate seminal plasma and a swim-up was performed to select the motile population which, in turn, was incubated up to 18 h at 37°C in 3.5% human serum albumin-supplemented Ham's F10 to allow for capacitation. For evaluation, sperm aliquots were taken pre-swim-up (T_o), immediately post swim-up (T₁), at 6 h (T₆), and 18 h (T₁₈) of incubation. The main outcome measures were computer-analyzed sperm motion parameters and hyperactivated motility, and immunodetection of phosphotyrosine (PY)-containing proteins. During the capacitating incubation, normozoospermic samples displayed maximum motility, velocity, and hyperactivation at T₆, significantly decreasing their values at T₁₈. PY-proteins were located both at the tail and head of spermatozoa. Their expression increased progressively during the incubation, being present in about 70% of the sperm tails at T₁₈. Asthenozoospermic samples showed an inability to respond to capacitation with an increase in motion parameters and PY-phosphorylation. At T₆, both hyperactivation and PY-phosphorylation were significantly lower than in normal samples. Our results suggest that PY-phosphorylation of tail proteins is highly conspicuous in human spermatozoa, and increases its incidence in a time-dependent manner, as more sperm become capacitated. Asthenozoospermic samples displaying low percentages of motile sperm and altered motion characteristics showed a decreased incidence of PY-phosphorelated sperm. Tail protein PY-phosphorylation may be related to sperm movement, especially to hyperactivated motility and its deficiency may be associated to asthenozoospermia.     

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.     

Calcitriol induces post-thawed bovine sperm capacitation

Background: Capacitation is a set of physiological changes sperms undergo to acquire fertilizing capacity. In vivo, this process is directly associated with high calcium levels in sperm cytoplasm. Calcitriol, the vitamin D hypercalcemic metabolite, is related to human sperm motility, capacitation, and acrosome reaction. This work aimed to study the effect of calcitriol on bull sperm quality parameters and capacitation. Methods: One million freeze-thawed spermatozoa were obtained from different bulls and treated with 20 nM of calcitriol for 30 min. Untreated cells (negative control) and treated ones with calcitriol or heparin (100 µg/mL, positive capacitation control) were evaluated for motility, viability, and functional parameters. Menadione (70 µM, 30 min) treatment was included as a reactive oxygen species (ROS) positive sperm agent. Results: The results elucidated that sperm exposed to 20 nM calcitriol showed higher viability, vigor, and capacitation than their positive and negative controls. The percentage of sperm with intact plasma and acrosome membranes, mitochondrial membrane potential (ΔΨm), and phosphatidylserine externalization was similar in all the conditions evaluated, while ROS production was higher with heparin and menadione-treated groups than the calcitriol group or negative control. Conclusion: Our results indicate that calcitriol induces the capacitation of thawed bull spermatozoa and maintains acceptable values of progressive motility, viability, and vigor without altering key biological parameters such as redox status, ΔΨm, and cell death.     

Effect of peroxiredoxin 6 on total and progressive motility of human spermatozoa after cryopreservation

Sperm cryopreservation is useful in assisted reproductive technology and male fertility preservation. However, freezing and thawing significantly reduces the total and progressive motility of human spermatozoa. In the present study, we explored the effects of peroxiredoxin 6 (PRDX6) on total and progressive motility of human spermatozoa after cryopreservation. Semen samples of 20 males with normal parameters were collected and frozen in media supplemented with different concentrations of PRDX6 (0 mM, 10⁻⁵ mM, 10⁻⁷ mM, and 10⁻⁹ mM, respectively). Postthaw total and progressive motility of sperms were measured. The results showed that in comparison with 0 mM, the concentrations of 10⁻⁵ mM, 10⁻⁷ mM, and 10⁻⁹ mM of PRDX6 all significantly improved total motility and progressive motility of sperms (p < 0.05). The 10⁻⁷ mM of PRDX6 showed the best performance. In conclusion, the supplementation of PRDX6 helps to maintain the total and progressive motility of human spermatozoa.     

A thermal study of cellular motility by optical time-resolved correlation

The study of motor properties of cells under appropriate physical-chemical conditions is a significant problem nowadays. The standard techniques presently used do not allow to evaluate neither large samples nor to control their thermodynamic conditions. In this work, we report a cell motility sensor based on an optical technique with a time-resolved correlation, adapted in a system able to study several samples simultaneously. Image correlation analysis is used to follow their temporal behavior. A wide variety of motile cells, such as archaea, bacteria, spermatozoa, and even contractile cells, can be studied using this technique. Here, we tested our technique with the study of sperm motility. In particular, both the sperm motility and its prevalence are studied under a temperature range from 0 to 37 °C. We found that incubation at 10 °C presents the lengthiest prevalence in motility and observed, for the first time, an interesting thermal reversibility behavior.     

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.     

Dispermic fertilization of human oocytes

Regardless of whether fertilization occurs in vivo or in vitro, polyspermic penetration of the human oocyte is a not infrequent cause of reproductive failure. This report describes the occurrence of human eggs fertilized by two spermatozoa and the variable developmental potentials expressed by the resultant embryos. The cellular and subcellular events that characterize the pronuclear and early cleavage stages of preimplantation embryogenesis are discussed with respect to the ability of such embryos to progress to implantation.     

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.     

Harmful effects of pyrethroid ester insecticide on the male reproductive system mainly through affecting testicular function and inflammatory markers

Pyrethroid esters are widely used as insecticides worldwide. In this study, we aimed to evaluate the harmful effect of deltamethrin on the male reproductive system through the assessment of reproductive hormones, inflammatory markers, and testicular function. To achieve our aim, eighty male 7-9-week-old, Wistar rats were taken, weighed, and divided into four experimental groups. The first group was kept as a control group, and the other three groups were given deltamethrin orally at different concentrations (0.87, 8.7, and 17.4 mg/kg body weight) for nine weeks. The results indicated that deltamethrin administration associated with a significant decrease in reproductive hormones, especially FSH, LH, and significant elevation in the interleukin 2 (IL2), interleukin 6 (IL6), histamine, and cortisol levels. Also, the significance of inhibition of sperm motility and viability, decreased testis weights, sperm count, and fructose in semen were noted. These findings clarify the harmful effect of deltamethrin on the male reproductive system by producing a significant alteration in reproductive hormones, inflammatory markers as well as testicular function.     

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.