Alcohol extract of Bauhinia forficata link reduces lipid peroxidation in the testis and epididymis of adult Wistar rats

Bauhinia forficata is a medicinal plant that has flavonoid components with hypoglycemic, antioxidant, hepatoprotective, antibacterial, antiviral and anti‐inflammatory action. Aim of this study is to evaluate the action of B. forficata alcoholic extract in the male genital system of adult male Wistar rats. For that, 20 adult male Wistar rats were distributed into two experimental groups: the B. forficata group, receiving B. forficata alcoholic extract (0.1 ml/10 g body weight/day) on alternate days, and the control group, receiving just the vehicle for 30 days straight both via gavage. On the 31st day, the animals were euthanized, and the testis and epididymis were collected for histopathological, biochemical, morphometric, and sperm count analysis. Mass spectrometry identified new compounds in the extract: trans‐caffeic acid, liquiritigenin, gallocatechin, and 2,4,6‐trihydroxyphenanthren‐2‐glycoside. Biochemical analysis showed higher total cholesterol levels in the testis and lower malondialdehyde levels in the testis and epididymis, in the B. forficata group. The mast cell count showed a reduction in degranulated mast cells in the caput region of the epididymis, in the B. forficata group. The luminal compartment of the caput and the epithelial of the epididymis cauda were reduced, whereas the stromal region of the epididymis caput was increased in the B. forficata group, compared with the control group. The testicular tissue was less impaired, considering that all the histological analyses were similar to the control. We believe that B. forficata alcoholic extract in the male genital system showed antioxidant action, especially in the epididymal tissue.     

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.     

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.     

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.     

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.     

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.     

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.     

Interaction of maternal separation on the UCh rat Cerebellum

Maternal care is the main source of signals and stimuli for proper development, growth, and production of adjustment responses to stressful factors. Adverse experiences in childhood are associated with a vulnerability to developing abusive ethanol ingestion via alterations of the response of the hypothalamic–pituitary–adrenal axis. Alcoholism causes global brain abnormalities, with the cerebellum being one of the most susceptible areas. We evaluated the effect of maternal separation on the cerebellum structure of male UCh rats. Adult male UChA (low 10% ethanol consumption) and UChB (high 10% ethanol consumption) rats were divided in to four experimental groups: (1) UChA, (2) UChA maternal separation (MS), (3) UChB, and (4) UChB MS. The MS occurred between the 4th and 14th days of age, for 240 min day^?1. Euthanasia was performed at 120 days of age. An image analysis system was used to measure cerebellar cortical height and Purkinje cellular area and height in five rats from each group. The cerebellar sections were stained with antibodies against IGFR‐I. MS did not alter the ethanol consumption of UChA and UChB rats. Corticosterone level was significantly higher in UChA MS and UChB MS rats than in UChA and UChB rats. The Purkinje cellular area and height were higher in UChA MS rats. IGFR‐I expression was observed in the cortical glomerular area of UChA MS and UChB MS rats. MS altered the Purkinje cells in the cerebella of male UCh rats. Microsc. Res. Tech. 77:44–51, 2014. © 2013 Wiley Periodicals, Inc.     

Contribution of epididymal secretory proteins for spermatozoa maturation

The final stages of sperm differentiation occur outside the gonad and are not under the genomic control of germ cells. Only sequential interactions with the medium surrounding the sperm are believed to induce the final steps of spermatogenesis. The epididymis, a long tubule with very active secretory and reabsorption functions, is able to create sequential changes in the composition of luminal fluid throughout its length. The chronologies of the changes, which occur on/in the sperm with those in their surrounding environment, are described. Correlations between the highly regionalized epididymal activities and sperm characteristics linked to their survival and fertility potential are presented in this review.     

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.     

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.     

Ketamine effects on the urogenital system--changes in the urinary bladder and sperm motility

Different doses of ketamine (10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, and 60 mg/kg) were injected i.p. (I.P.), respectively, to male ICR mice to determine the optimal dosage for chronic administration. At and above 40 mg/kg I.P. injection, mice had almost no hindlimb movement during swimming test. Subsequently, 30 mg/kg was used as the dose for the study in the toxicity of long-term ketamine administration on urinary bladder and sperm motility. The treatment group were subdivided into two (n = 10 each group); one received daily ketamine treatment i.p. for 3 months and another group for 6 months. Corresponding number of mice in control groups (n = 5 each group) received saline injection instead of ketamine. Terminal dUTP nick and labeling (TUNEL) study and Sirius red staining were carried out on the sectioned slides of the urinary bladders to study the degree of apoptosis in both epithelium and muscular layers of the urinary bladder and the relative thickness of the muscular layers in this organ was also computed. Apoptosis in the bladder epithelium was observed initially in the 3-month ketamine treated mice and the number of apoptotic cells was significantly different (P < 0.05) between the 3-month and 6-month ketamine treated mice and the control. The relative thickness of muscular layers in the bladder wall also decreased significantly (P < 0.05) when the 6-month treated mice and the control were compared. Sirius red staining revealed increase of collagen in the urinary bladder of the treated mice, most evidently 6 months after ketamine treatment. In addition, the sperm motility was studied and there was a statistically significant difference between the control and ketamine treated groups in the percentages of sperms which were motile (P < 0.05). This suggested that the chronic administration of ketamine affected the genital system as well.     

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.     

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.     

Role of resistance physical exercise in preventing testicular damage caused by chronic ethanol consumption in UChB rats

Ethanol consumption is associated with spermatogenesis damage and testosterone level alterations. Alcohol remains the most commonly used substance among athletes and sports enthusiasts. This study evaluated whether resistance physical exercise can reduce the testicular damage caused by ethanol exposure. A total of 36 ethanol drinking (UChB) rats were divided into four groups: C (control rats), ETOH (ethanol consumption), ETOH + T (ethanol consumption + physical training), and T (group physical training). The physical training component of the T and ETOH + T groups was based on a resistance training model consisting of four sets of 10 jumps, with an increasing overload of 50–70% of the body weight attached to the chest three times per week. Rats in the ETOH and ETOH +T groups received 10% ethanol. At postnatal day 90, the rats were sacrificed. Blood sample was collected for hormonal analysis, and the testicles were weighed and processed for histopathological, morphometric, and immunohistochemical analyses. The ETOH group showed an increase in testosterone levels. The immunohistochemical of androgen receptor and the absolute weight of the testes were higher in the ETOH and ETOH + T groups, while the ETOH animals showed a decreased weight gain index. The number of abnormal seminiferous tubules increased in the ETOH and T groups compared to those in the control group (C); however, the association with treatment (ETOH + T group) prevented this effect and decreased caspase‐3 production. In conclusion, these findings show that the combination of ethanol consumption and resistance physical exercise can prevent testicular damage in adult UChB rats.     

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.     

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.     

Advances in cryopreservation of spermatogonial stem cells and restoration of male fertility

Spermatogenesis is a highly complicated process which initiated by spermatogonial stem cells (SSCs). SSCs are the only cell type that can restore fertility in infertile recipient after SSCs transplantation. SSCs damage during cancer diagnosis and therapy and their depletion may be cause of male infertility in cancer survivors. In this review, used experimental methods regarding SSCs and testis tissue cryopreservation have been reviewed with a special focus on animal models and human which have generated the majority of data about SSCs and the cryopreservation process. Microsc. Res. Tech. 79:122–129, 2016. © 2015 Wiley Periodicals, Inc.     

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.