FASCIOLA-HEPATICA - DISRUPTION OF SPERMATOGENESIS BY THE MICROFILAMENT INHIBITOR CYTOCHALASIN-B

The distribution of actin filaments in the spermatogenic cells of Fasciola hepatica was determined using a fluorescent derivative of phalloidin. Actin was localised primarily in the region of separation of a secondary spermatogonium from a primary spermatogonium, in the inner faces at the centre of four-cell clusters of tertiary spermatogonia and in the cytophore region of spermatocyte and spermatid rosettes. The effect of the microfilament inhibitor cytochalasin B (100-mu-g/ml) on the ultrastructure of the spermatogenic cells was determined in vitro by transmission electron microscopy using tissue-slice material. Cytochalasin B treatment led to the formation of bi- and multinucleate cells, whose frequency increased with progressively longer incubation periods. Few typical rosettes of spermatocyte and spermatid cells were evident from 6 h onwards, being replaced by syncytial masses of cells. Spermatozoon formation became abnormal in the longer treatment periods, the spermatozoa containing variable numbers of axonemes and an altered distribution of cortical microtubules. Multiple axonemes were observed in the cytoplasm of spermatid cells. The results are discussed in relation to the established role of actin in the cytokinesis phase of cell division and to the effects of cytochalasin B on other tissues and organ systems within the fluke.

Fasciola hepatica: A light and electron microscope study of sustentacular tissue and heterophagy in the testis

In order to investigate cytolytic activity in the testis of Fasciola hepatica, flukes belonging to several different triclabendazole (TCBZ)-sensitive and TCBZ-resistant isolates, and wildtype flukes from field infections, were studied by light and electron microscopy with a view to identifying sites of heterophagy and macromolecular hydrolysis. At the periphery of the testis tubules in all the flukes examined, large euchromatic nuclei, each bearing a prominent nucleolus, were seen. These were invested with a thin cytoplasmic layer, extensions of which partially enveloped and probably supported the neighbouring spermatogonia. No lateral cell boundaries were identified in this tissue, possibly indicating syncytial organisation. The tissue, considered to be analogous to Sertoli cells in vertebrate testis, was identified as sustentacular tissue. It displayed cytoplasmic features consistent with protein/glycoprotein synthesis (through a granular endoplasmic reticulum-Golgi mediated mechanism) and intracellular digestion/heterophagy (through a lysosomal system). The intracytoplasmic hydrolytic activity of the sustentacular tissue probably serves to scavenge effete cells and cytoplasmic debris, to recycle useful molecules, to promote spermatozoon maturation and possibly to aid osmoregulation within the tubules. Certain protein-containing macromolecules synthesised in the sustentacular tissue may contribute to the seminiferous fluid, or have pheromonal activity. The presence of numerous mitochondria and abundant smooth endoplasmic reticulum is consistent with facilitation of inward and outward movement of micromolecular nutrients, metabolites including excretory products and water. In the sustentacular tissue of certain flukes with dysfunctional spermiogenesis, there was increased heterophagic and cytolytic scavenging activity. The cytoplasmic residual vacuoles remaining after the release of spermatids were also identified as possible sites for lysosome-mediated intracellular digestion and osmoregulation in the testis tubules of F. hepatica. (C) 2012 Elsevier B.V. All rights reserved.

Fasciola hepatica:disruption of spermatogenesis by the microfilament inhibitor cytochalasin B

The distribution of actin filaments in the spermatogenic cells of Fasciola hepatica was determined using a fluorescent derivative of phalloidin. Actin was localised primarily in the region of separation of a secondary spermatogonium from a primary spermatogonium, in the inner faces at the centre of four-cell clusters of tertiary spermatogonia and in the cytophore region of spermatocyte and spermatid rosettes. The effect of the microfilament inhibitor cytochalasin B (100 micrograms/ml) on the ultrastructure of the spermatogenic cells was determined in vitro by transmission electron microscopy using tissue-slice material. Cytochalasin B treatment led to the formation of bi- and multinucleate cells, whose frequency increased with progressively longer incubation periods. Few typical rosettes of spermatocyte and spermatid cells were evident from 6 h onwards, being replaced by syncytial masses of cells. Spermatozoon formation became abnormal in the longer treatment periods, the spermatozoa containing variable numbers of axonemes and an altered distribution of cortical microtubules. Multiple axonemes were observed in the cytoplasm of spermatid cells. The results are discussed in relation to the established role of actin in the cytokinesis phase of cell division and to the effects of cytochalasin B on other tissues and organ systems within the fluke.

Anàlisi microscòpica de l'esperma ejaculada i de la maduració epididimària dels espermatozoides de Sus domesticus

El present treball analitza la morfologia espermàtica de l'ejaculat de Sus domesticus, la histologia del conducte epididimari i la qualitat de l'esperma epididimari. El material d'estudi prové de mascles reproductors porcins de les races Landrace i Pietrain, sans i sexualment madurs. La metodologia emprada es basa en l'examen al microscopi òptic (camp dar, contrast de fases i contrast interferencial) i al microscopi electrònic (de rastreig i de transmissió). Per a l'anàlisi estadística de les dades s'ha utilitzat el test de la X2 de Pearson (p<0,01). L'estudi de la morfologia espermàtica de l'ejaculat permet distingir diversos tipus de gàmetes que s'han classificat en tres grups: espermatozoides madurs, espermatozoides immadurs i espermatozoides aberrants, així com algunes cèI.lules somàtiques. L'espermatozoide madur de Sus domesticus és un gàmeta típic de mamífer (format per tres parts: cap, peça de connexió i cua) en que destaquen: la forma oval i plana del cap, el desenvolupament d'una protuberància acrosòmica apical en una de les cares del cap i la presencia dels cossos laminars en la peça de connexió. L'espermatozoide immadur es caracteritza per la presencia de la gota citoplasmàtica, el major desenvolupament de la protuberància acrosòmica apical i per la flexibilitat del cap. Els espermatozoides aberrants es descriuen i classifiquen segons la morfologia externa i la morfologia interna, distingint-se una amplia gama de malformacions que afecten les diverses parts de l'espermatozoide. Les cèl·lules somàtiques presents en l'ejaculat ofereixen les característiques pròpies d'un macròfag i se les ha observat englobant espermatozoides immadurs. L'estudi de l'estructura i la ultraestructura de les tres regions anatòmiques de l'epidídim (caput, corpus i cauda) revela que: a) l'epiteli epididimari és pseudoestratificat amb esterocilis, b) cada regió epididimària presenta uns valors característics en relació al diàmetre intern del conducte, a l'alçada de l'epiteli, a la longitud dels esterocilis i al nombre de cèl·lules somàtiques luminals, i c) l'epiteli epididimari esta format per cinc tipus cel·lulars: les cèl·lules principals, les cèl·lules basals, les cèl·lules dares, les cèl·lules estretes i les cèl·lules basòfiles. Dels resultats obtinguts es pot deduir que: a) aquests cinc tipus cel·lulars es distribueixen al llarg del conducte epididimari de forma no homogènia, b) les cèl·lules basals, les cèl·lules principals, les cèI.Iules dares i les cèl·lules estretes són diversos estadis del desenvolupament d'un mateix tipus cel·lular especialitzat en la secreció i reabsorció cel·lular, i c) les cèl·lules basòfiles són les precursores de les cèl·lules somàtiques luminals. La qualitat de l'esperma procedent de les tres regions de l'epidídim ha estat analitzada a partir dels següents paràmetres espermàtics: vitalitat, resistència osmòtica dels acrosomes, estabilitat cefàlica, morfologia, malformacions i aglutinació. La vitalitat espermàtica disminueix progressivament al llarg del conducte epididimari. La resistència osmòtica dels acrosomes s'assoleix en la regió corporal de l'epidídim. L'estabilitat cefàlica dels espermatozoides és més elevada en les dues primeres regions de l'epidídim que en la regió caudal. Cada regió de l'epidídim es caracteritza per una morfologia espermàtica específica: a) el caput es caracteritza per l'elevat percentatge d'espermatozoides immadurs amb gota citoplasmàtica proximal, b) el corpus es caracteritza per l'elevat percentatge d'espermatozoides immadurs amb gota citoplasmàtica distal, i c) el cauda es caracteritza per l'elevat percentatge d'espermatozoides madurs. S'han estudiat les següents malformacions d'origen epididimari: espermatozoides de cua doblegada per l'anell de Jensen (origen en el cauda), espermatozoides de cua enrotllada i espermatozoides de cues fusionades (origen en el corpus). Els espermatozoides perden la capacitat de doblegar la cua per la peça intermèdia a mesura que avancen pel conducte epididimari. L'aglutinació espermàtica tendeix a augmentar progressivament al llarg del conducte epididimari, si bé, no s'han observat variacions significatives en els diversos tipus d'aglutinació. La maduració epididimaria dels espermatozoides de Sus domesticus és un procés lent i complex, i la qualitat de l'ejaculat depèn de que aquesta maduració hagi estat completa. La presencia en l'esperma ejaculat de formes gamètiques pròpies de l'esperma epididimari és un signe d'una incompleta maduració dels espermatozoides; i, pot considerar-se com un paràmetre indicador d'estrés del mascle reproductor, tant més quant més s'assembli a la morfologia espermàtica de la regió cefàlica de l'epidídim.

Spermatogenesis in the blue swimming crab, Portunus pelagicus, and evidence for histones in mature sperm nuclei

Spermatogenesis in the blue swimming crab, Portunus pelagicus, is described by light and electron microscopy. The testis is composed of anterior (AT) and posterior (PT) lobes, that are partitioned into lobules by connective tissue trabecula, and further divided into zones (germinal, transformation and evacuation), each with various stages of cellular differentiation. The vas deferens is classified into three distinct regions: anterior (AVD), median (MVD), and posterior (PVD), on the presence of spermatophores and two secretions, termed substance I and II. Based on the degree and patterns of heterochromatin, spermatogenesis is classified into 13 stages: two spermatogonia (SgA and SgB), six primary spermatocytes (leptotene, zygotene, pachytene, diplotene, diakinesis, and metaphase), a secondary spermatocyte (SSc), three spermatids (St 1–3), and a mature spermatozoon. Spermatid stages are differentiated by chromatin decondensation and the formation of an acrosomal complex, which is unique to brachyurans. Mature spermatozoa are aflagellated, and have a nuclear projection and a spherical acrosome. AUT-PAGE and Western blots show that, during chromatin decondensation, there is a reduction of most histones, with only small amounts of H2B and H3 remaining in mature spermatozoa.

Ultrastructural analysis of spermiogenesis in the neotropical cichlid Cichla kelberi Kullander & Ferreira, 2006 (Perciformes: Cichlidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structural analysis of fertilization in the fish Brycon orbignyanus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Significance of extruded nuclear chromatin (regional nuclear shape malformation) in human spermatozoa: implications for ICSI

The aim of this study was to determine the extent of DNA fragmentation and the presence of denatured single-strand or normal double-strand DNA in spermatozoa with extruded nuclear chromatin (ENC) selected by high magnification. Fresh semen samples from 55 patients were prepared by discontinuous isolate concentration gradient. Spermatozoa with normal nucleus (NN) and ENC were selected at 8400x magnification and placed on different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double-stranded DNA was identified by the acridine orange fluorescence method. DNA fragmentation was not significantly different (p = 0.86) between spermatozoa with ENC (19.6%) and those with NN (20%). However, the percentage of spermatozoa with detectable denatured-stranded DNA in the ENC spermatozoon group (59.1%) was significantly higher (p < 0.0001) than in the NN group (44.9%). The high level of denatured DNA in spermatozoa with ENC suggests premature decondensation and disaggregation of sperm chromatin fibres. The results show an association between ENC and DNA damage in spermatozoa, and support the routine morphological selection and injection of motile spermatozoa at high-magnification intracytoplasmic sperm injection.

Motile sperm organelle morphology examination (MSOME): intervariation study of normal sperm and sperm with large nuclear vacuoles

Background: Although the motile sperm organelle morphology examination (MSOME) was developed only as a selection criterion, its application as a method for classifying sperm morphology may represent an improvement in evaluation of semen quality, with potential clinical repercussions. The present study aimed to evaluate individual variations in the motile sperm organelle morphology examination (MSOME) analysis after a time interval.Methods: Two semen samples were obtained from 240 men from an unselected group of couples undergoing infertility investigation and treatment. Mean time interval between the two semen evaluations was 119 +/- 102 days. No clinical or surgical treatment was realized between the two observations. Spermatozoa were analyzed at greater than or equal to 8400 x magnification by inverted microscope equipped with DIC/Nomarski differential interference contrast optics. At least 200 motile spermatozoa per semen sample were evaluated and percentages of normal spermatozoa and spermatozoa with large nuclear vacuoles (LNV/one or more vacuoles occupying >50% of the sperm nuclear area) were determined. A spermatozoon was classified as morphologically normal when it exhibited a normal nucleus (smooth, symmetric and oval nucleus, width 3.28 +/- 0.20 mu m, length 4.75 +/- 0.20 mu m/absence of vacuoles occupying >4% of nuclear area) as well as acrosome, post-acrosomal lamina, neck and tail, besides not presenting cytoplasm around the head. One examiner, blinded to subject identity, performed the entire study.Results: Mean percentages of morphologically normal and LNV spermatozoa were identical in the two MSOME analyses (1.6 +/- 2.2% vs. 1.6 +/- 2.1% P = 0.83 and 25.2 +/- 19.2% vs. 26.1 +/- 19.0% P = 0.31, respectively). Regression analysis between the two samples revealed significant positive correlation for morphologically normal and for LNV spermatozoa (r = 0.57 95% CI: 0.47-0.65 P < 0.0001 and r = 0.50 95% CI: 0.38-0.58 P < 0.0001, respectively).Conclusions: The significant positive correlation and absence of differences between two sperm samples evaluated after a time interval with respect to normal morphology and LNV spermatozoa indicated that MSOME seems reliable (at least for these two specific sperm forms) for analyzing semen. The present result supports the future use of MSOME as a routine method for semen analysis.

Características ultra-estruturais e diferenciativas das espermátides de piracanjuba (Brycon orbignyanus) durante a espermiogênese

A espermiogênese que ocorre em piracanjuba Brycon orbignyanus pode ser dividida em quatro etapas morfológicas, cujas características principais consistem em reduções dos volumes citoplasmático, nuclear e celular, e compactação da cromatina nuclear das espermátides, sendo que as etapas espermiogenéticas ocorrem simultaneamente. Ao final da espermiogênese, quando as espermátides atingem nível elevado de diferenciação, os núcleos se tornam mais compactos e os citoplasmas se tornam reduzidos. Estas modificações resultam na formação de células altamente diferenciadas, os espermatozóides com cabeça, peça intermediária e flagelo bem definidos. As espermátides e os espermatozóides foram observados em cistos germinativos, mas também podem ser encontrados na luz dos túbulos seminíferos.

Ultrastructural study of acrosome formation in mongolian gerbil (Meriones unguiculatus)

Spermatogenesis is a complex and very well organized process lasting from 30 to 75 days in mammals. The spermatogenic process has been described mainly in laboratory mammals, such as the rat, while correspondent studies in wild animals are scarce. The gerbil (Meriones unguiculatus) is a small rodent native of the arid regions of Mongolia and China. Few reports are available on reproduction in the male Mongolian gerbil. The present study provides the first description of the ultrastructural alterations in spermatid cytoplasm and nucleus, with particular reference to acrosome formation in gerbils. The testes were processed by conventional transmission electron microscopy technique. Based on the development of the acrosomal system and changes in nuclear morphology, the transformation of spermatids in spermatozoon was divided into 15 steps. There were four phases in the spermiogenesis process in the gerbil: Golgi, cap, acrosomal and maturation phases. This provides the foundation for a variety of future studies of the spermiogenesis of this animal. (C) 2000 Harcourt Publishers Ltd.

Ultrastructure of spermiogenesis and spermatozoa of Gymnotus cf. anguillaris and Brachyhypopomus cf. pinnicaudatus (Teleostei : Gymnotiformes)

The ultrastructure of spermiogenic stages and spermatozoa of representatives of two gymnotiform families, Gymnotus cf. anguillaris (Gymnotidae) and Brachyhypopomus cf. pinnicaudatus (Hypopomidae) were studied. Spermiogenesis of both species is characterized by lateral development of the flagellum and formation of a nuclear fossa. Some differences were found between these species, such as whether (B. cf. pinnicaudatus) or not (G. cf. anguillaris) nuclear rotation occurs, permanence of the cytoplasmic channel, and type and localization of the nuclear fossa. In the G. cf anguillaris spermatozoon the nucleus is spherical with highly condensed chromatin. The nuclear fossa is shallow and lateral and is associated with the centriolar complex through stabilizing fibrils. The midpiece is short, with many vesicles, a cytoplasmic channel, and elongate mitochondria. In the B. cf. pinnicaudatus spermatozoon the ovoid nucleus is elongated lateral and posterior to the centriolar complex, and has highly condensed chromatin. The eccentric nuclear fossa is of the moderate type, and contains the entire centriolar complex. The midpiece is long, with numerous vesicles, elongate mitochondria, and no cytoplasmic channel. In both species the flagella are laterally disposed in relation to the nucleus and comprise of the classical 9 + 2 axoneme. Most of the characteristics found in the spermatozoa of these two species of Gymnotiformes are shared with species of Characiformes, whereas only a few are also found in Siluriformes. This suggests that Gymnotiformes and Characiformes may be more closely related than previously proposed. (C) 2007 Elsevier Ltd. All rights reserved.

Ultrastructure of spermiogenesis in Plagioscion squamosissimus (Teleostei, Perciformes, Sciaenidae)

Spermiogenesis in Plagioscion squamosissimus occurs in cysts. It involves a gradual differentiation process of spermatids that is characterized mainly by chromatin compaction in the nucleus and formation of the flagellum, resulting in the spermatozoa, the smallest germ cells. At the end of spermiogenesis, the cysts open and release the newly formed spermatozoa into the lumen of the seminiferous tubules, the spermatozoa do not have an acrosome and are divided into head, midpiece, and tail or flagellum, the spermatozoa of P. squamosissimus are of perciform type with the flagellum parallel to the nucleus and the centrioles located outside the nuclear notch. (C) 1999 Harcourt Publishers Ltd.

Ultrastructure of spermatogenic cells and spermatozoa in Hoplias malabaricus (teleostei, characiformes, erythrinidae)

The differentiation of spermatids in Hoplias malabaricus is characterized by chromatin compaction, flagellum development, nuclear rotation, nuclear fossa formation, and excess cytoplasm elimination. In the resulting spermatozoon, the head is round and the nucleus contains chromatin compacted in thick filaments, peripherically arranged, to a central electron-lucent area. The acrosome is absent. The nuclear fossa is eccentric but not pronounced. The proximal centriole penetrates it and is oblique to the flagellum. The long midpiece has several converging elongate vesicles, forming membranous hoops in the initial segment of the flagellum, but has no cytoplasmic channel. The mitochondria are elongate and branched or C-shaped and located around the initial segment of the axoneme. The lateral flagellum does not show lateral projections. The ultrastructural characteristics of H. malabaricus spermatozoa are similar to the Cypriniformes. (C) 2001 the Fisheries Society of the British Isles.

The ultrastructure of spermiogenesis and spermatozoa in Diplomystes mesembrinus

Spermiogenesis in Diplomystes mesembrinus, one of the most primitive species from the Siluriformes, occurs in cysts. Differentiation of spermatids is characterized by chromatin compaction, flagellum development, nuclear fossa formation, rotation of the nucleus, and excess cytoplasm elimination. The spermatozoon head is round, the nucleus contains highly condensed chromatin clusters, the midpiece is short, the axoneme shows a 9 + 2 pattern with two discrete lateral projections, and the acrosome is absent. The nuclear fossa penetrates deeply into the nucleus, including the centriolar complex and the start of the axoneme. The single large C-shaped mitochondrion sur rounds the: initial segment of the axoneme. The structural features of D. mesembrinus spermatozoon are similar to the Clupeiformes. (C) 2001 the Fisheries Society of the British Isles.