Histochemical Approaches Research Articles

Novel Fluorescence-Based Methods to Determine Infarct and Scar Size in Murine Models of Reperfused Myocardial Infarction.

Determination of infarct and scar size following myocardial infarction (MI) is commonly used to evaluate the efficacy of potential cardioprotective treatments in animal models. However, histological methods to determine morphological features in the infarcted heart have barely improved since implementation while still consuming large parts of the tissue and offering little options for parallel analyses. We aim to develop a new fluorescence technology for determining infarct area and area at risk that is comparable to 2,3,5-triphenyltetrazolium chloride (TTC) staining but allows for multiple analyses on the same heart tissue. For early and late time points following MI, we compared classical histochemical approaches with fluorescence staining methods. Reperfused MI was induced in male mice, the hearts were extracted 24 h, 7-, 21-, or 28-days later and fluorescently stained by combining Hoechst and phalloidin. This approach allowed for clear visualization of the infarct area, the area at ischemic risk and the remote area not affected by MI. The combined fluorescence staining correlated with the classic TTC/Evans Blue staining 24 h after MI (r = 0.8334). In later phases (>7 d) post-MI, wheat germ agglutinin (WGA) is equally accurate as classical Sirius Red (r = 0.9752), Masson's (r = 0.9920) and Gomori's Trichrome (r = 0.8082) staining for determination of scar size. Additionally, feasibility to co-localize fluorescence-stained immune cells in specific regions of the infarcted myocardium was demonstrated with this protocol. In conclusion, this new procedure for determination of post-MI infarct size is not inferior to classical TTC staining, yet provides substantial benefits, including the option for unbiased software-assisted analysis while sparing ample residual tissue for additional analyses. Overall, this enhances the data quality and reduces the required animal numbers consistent with the 3R concept of animal experimentation.

Characteristics of the Integument of Phyllomedusa burmeisteri (Anura, Phyllomedusidae) and Boana semilineata (Anura, Hylidae) Males Can Contribute to Understanding its Relation to Habitat.

The integument of anurans plays vital physiological roles, crucial for understanding the species' survival in their environment. Despite its significance, there are few studies describing the cutaneous morphology of anurans from the Brazilian Atlantic Forest. This study aimed to characterize the integument of Phyllomedusa burmeisteri and Boana semilineata in males using microscopic and histochemical approaches. Histological sections were stained with various dyes, and additional fragments underwent electron microscopy and energy-dispersive X-ray spectroscopy. Results showed different projections on the dorsal and ventral regions of males from these species, without the Eberth-Katschenko layer. Differences in the arrangement of chromatophore cells in regions with varying solar incidence were observed in the spongy dermis. Various gland types were identified, aiding taxonomic differentiation and validation of behavioral data. Both species had seromucous and granular glands, while only P. burmeisteri displayed lipid glands. Histochemical analysis revealed higher production of polysaccharides and proteins, contributing to the integument's moisture and protection. Lipid secretions in P. burmeisteri helped waterproof the integument more effectively against desiccation. This study concludes that analyzing anuran integument provides valuable insights into their behavior, with integument composition potentially influenced by habitat choice among different species.

Morphoanatomical and histochemical investigation of Monteverdia evonymoides (Reissek) Biral

Monteverdia species are known for their medicinal properties. However, due to similarities between species, the accurate identification of different species can be cumbersome. The study of morphoanatomy can contribute to the authentication of the species by providing additional information and contributing to the species characterization. Therefore, this work presents the morphoanatomy of the leaves and stems of Monteverdia evonymoides (Reissek) Biral analyzed by optical microscopy, scanning electron microscopy, and histochemical approaches. Through these methods, we were able to determine, morphologically, the analyzed species features large and woody trees; its leaves are simple, distichous, and entire; the leaf margins are fringed; the stipules are caducous, triangular, and rarely persistent; the apex can vary from acute to obtuse, and the leaf blade has no trichomes. The frontal view presents polygonal epidermal cells, anomocytic, anisocytic, and tetracytic stomata that are found on the abaxial surface and are positioned at the same level as the ordinary epidermal cells; the epidermis is uniseriate and covered with a thick cuticle; the mesophyll is dorsiventral; the midrib is biconvex, and below the epidermis is the angular collenchyma, with one collateral bundle in an open arc and two dorsal bundles; in the ground parenchyma, there is a small collateral vascular bundle, with fibers juxtaposed to the phloem. Histochemical reactions were positive for lipophilic, phenolic, and lignified compounds. Among the observed characteristics, the stomatal classification of the leaf and the prominence of the midrib (adaxial surface) in this study help to identify M. evonymoides and may contribute to species taxonomy by comparing it with other species of the genus and quality control studies.

Anatomy and Histochemistry of Alliaceae and Iridaceae Species: Quality Control of Traditional Medicine Raw Materials

Alliaceae (Allium cepa and Allium ascalonicum) and Iridaceae (Eleutherine bulbosa) are widely used as spices, condiments, and traditional medicine. These three species are vulnerable to adulteration because they are morphologically similar in shape and colour. This study aims to identify and authenticate the three samples through anatomical and histochemical approaches. Anatomical and histochemical analyses were performed on fresh sections of the bulbs samples according to standard anatomical procedures with a razor blade and identification of the main compound groups using specific reagents. Anatomically, the three samples had similar patterns and cell organelles (cell nucleus, cytoplasm, and cell wall). Histochemical techniques identified the presence of alkaloids, phenolics, lipophilics, and terpenoids groups. Histochemical techniques can be applied in identifying the compound groups of the three samples and can be used as an alternative in testing the quality control of traditional medicine raw materials.

1954-2024: 70 years of histochemical research with the European Journal of Histochemistry.

This Editorial celebrates the 70th anniversary of the European Journal of Histochemistry since its foundation as Rivista di Istochimica Normale e Patologica, and introduces a Special Collection of selected articles on the application of the histochemical approach for investigating cell biological features and processes in animals and plants, and under diseased conditions. The year 2024 is a special one for histochemists, as 100 years ago J.W. Robert Feulgen and H. Rossenbeck introduced the histochemical procedure for the specific stoichiometric staining of DNA in histological samples: to commemorate this influential publication, three papers in the present issue are devoted to the application of the Feulgen reaction at light and electron microscopy, and in cytometry.

Effect of pyriofenone on the infection processes and cytological features of Blumeria graminis on wheat leaves.

Pyriofenone demonstrates outstanding efficacy in controlling powdery mildew. We investigated the impact of pyriofenone on the infection processes and cytological features of Blumeria graminis f. sp. tritici on wheat leaves. The preventive application of pyriofenone before inoculation did not inhibit conidial germination but effectively suppressed both appressorial and haustorial formation. Notably, haustorial formation was effectively inhibited, resulting in the complete suppression of successive lesion development and sporulation. Curative application of pyriofenone after inoculation also inhibited lesion expansion and sporulation. Furthermore, it had considerable impact on the morphogenesis of powdery mildew fungus. We observed multi-formed secondary appressoria, shrunken or bifurcated hyphae, abnormal conidiophores, and clubbed conidia-like structures. Subsequently, we employed a histochemical approach to analyze the localization of essential components for the polar growth of fungal hyphae. Pyriofenone induced mislocalization of the actin cytoskeleton, β-glucan and cytoplasmic vesicles, although it did not affect tubulin orientation.

Flow cytometric detection of CD11b+ Gr-1+ cells in nontumor-bearing mice: A propolis-elicited model.

Myeloid-derived suppressor cells (MDSCs) are a heterogenous myeloid lineage population whose conventional surface phenotype is CD11b+ Gr-1+. Due to their rarity and fragility, analyses using primary isolated MDSCs are extremely difficult. However, counting CD11b+ Gr-1+ cells in associated tissues such as tumors and inflammatory lesions provides critical information regarding MDSC involvement in immune disorders in the tissues. Specific MDSC markers have not been identified, limiting our ability to apply histochemical approaches during MDSCs research. However, profiling surface antigens using multi-colorimetric flow cytometry enables us to easily monitor the abundance of MDSCs in vivo. Monitoring of mouse MDSCs and their subpopulations using flow cytometry is well established. In this article, I exemplify a conventional method of monitoring CD11b+ Gr-1+ cells in mouse adipose tissue after administration of Brazilian propolis ethanol extract, which is a strong inducer of MDSCs.

Morphological and cellular organization of green microalgae to cope with cold stress in subarctic environment

Microalgae are one of the most widely dispersed living organisms on Earth and can be found even in extreme environments. Especially in such habitats, the microalgal cell wall plays an essential role as it is the first barrier in continuous contact with the surrounding and changing environment. In cold conditions microalgae can show changes in their morphology, also known as phenotypic plasticity which is the ability of an organism to show different phenotypes when exposed to different environmental conditions. In addition, presence or absence of algaenan is thought to be responsible for increase/decrease permeability and stiffness of the cell wall. The aim of this work was to evaluate and compare how microalgae cells can modify their cell wall components and cellular morphology under low-temperature conditions (5 °C) and differ during the exponential and stationary phases. Four microalgae species were studied: Coelastrella sp. 3–4, Chlorella vulgaris sp. 13–1, Haematococcus pluvialis, and Scenedesmus sp. B2–2, which were isolated from subarctic locations.Using a histochemical approach in conjunction with light microscopy, cell features such as size, organization and cell wall ornamentation were evaluated. Staining procedures showed changes in biochemical components such as pectins and presence or absence of exopolysaccharides and lipids. Results showed that Coelastrella cultures did not grow under low-temperature conditions. However, Chlorella vulgaris, Haematococcus pluvialis and Scenedesmus species demonstrated a slower growth rate, bigger and rounded cell-shape during cold condition. Furthermore, the latter microalgal strain also showed modification in algaenan presence as one of the main components in cell wall architecture, which can be related to the permeability of cell wall. Changes in other features such as cell organization and cell wall ornamentation were investigated.

The intrinsically disordered C-terminus of purinoceptor P2K1 fine-tunes plant responses to extracellular ATP.

P2K1 is a plant-specific purinoceptor that perceives extracellular ATP (eATP), a signaling molecular implicated in various physiological processes. Interestingly, P2K1 harbors a C-terminal intrinsically disordered region (IDR). When we overexpressed a truncated P2K1 (P2K1t ) lacking the IDR, primary root growth completely ceased in response to eATP. We investigated the functional roles of the IDR in P2K1 using a combination of molecular genetics, calcium imaging, gene expression analysis, and histochemical approaches. We found that the P2K1t variant gave rise to an amplified response to eATP, through accumulation of superoxide, altered cell wall integrity, and ultimate cell death in the primary root tip. Together, these observations underscore the significant involvement of the C-terminal tail of P2K1 in root growth regulation.

Structural and histochemical approach to the fruit and seed diversity of Cyperaceae in an evolutionary context.

The Cyperaceae fruit consistency depends on the mesocarp. Seed structure is diverse and related to the evolutionary history of their species. A new storage tissue is described for Cyperaceae. Anatomy and histochemistry of Cyperaceae fruits (including seeds) are poorly known due to their hard, isolating tissues that prevent anatomical techniques. We performed the first, most comprehensive structural diversity characterisation of fruit and seed in Cyperaceae, accompanied by an unprecedented histochemical characterisation of seeds for this family. We analysed fruits of 29 species, included in 19 genera and 12 tribes within the subfamilies Cyperoideae and Mapanioideae, using light microscopy. Cyperaceae fruits have a pericarp with a one-cell-layered exocarp and endocarp, and a multi-cell-layered mesocarp. The mesocarp of the Mapanioideae has a spongy-fleshy outer region and a hard inner region. The mesocarp of the Cyperoideae has only a hard region. The pericarp is free from the seed coat. Cyperaceae seeds have a three-layered seed coat, an embryo with haustorial function of its scutellum, and two storage tissues: the endosperm and a putative perisperm. Nine seed morphotypes and four seed subtypes were observed among the studied species. Our results suggested that the fruit consistency is determined by the mesocarp. Both the terms "nut" and "achene" should be accepted to refer to the dry fruit of the Cyperaceae until a widely accepted fruit classification for angiosperms is proposed. The Cyperaceae seed structural diversity is high and related to the evolutionary history of the species. The "perisperm" is a new tissue proposed for sedge seeds, and is here characterized for the first time. The seed coat has a different structure than the one described so far for the family.

Morphological Analysis of Aging Hallmarks in the Central Nervous System of the Fast-Aging African Turquoise Killifish.

As the number of elderly individuals is increasing in modern society, the need for a relevant gerontology model is higher than ever before. Aging can be defined by specific cellular hallmarks, described by López-Otín and colleagues, who provided a map which can be used to scavenge the aging tissue environment. As revealing the presence of individual hallmarks does not necessarily indicate aging, here we provide different (immuno)histochemical approaches that can be used to investigate several aging hallmarks-namely, genomic damage, mitochondrial dysfunction/oxidative stress, cellular senescence, stem cell exhaustion, and altered intercellular communication-in the killifish retina, optic tectum, and/or telencephalon at a morphological level. In combination with molecular and biochemical analysis of these aging hallmarks, this protocol offers the opportunity to fully characterize the aged killifish central nervous system.

Thymol Induces Cell Death of Fusarium oxysporum f. sp. niveum via Triggering Superoxide Radical Accumulation and Oxidative Injury In Vitro

Fusarium oxysporum f. sp. niveum (FON) causes watermelon wilt that is one of the major disease-causing yield losses of watermelon. Sustainable development of agriculture requires controlling watermelon wilt disease with good environmental performance. One important approach is to identify environmental-friendly compounds with inhibitory activity against FON. Thymol is a plant-derived compound that is safe for ecology. Little is known about the application of thymol in agriculture. In this study, we studied the inhibitory activity of thymol against FON by using morphological, physiological, and histochemical approaches. Thymol significantly inhibited colony diameter of FON in a dose-dependent manner, with EC50 at 21 µg/mL. Thymol at 10, 21, and 35 µg/mL decreased the fresh weight of FON mycelia by 29.0%, 50.6%, and 69.5%, respectively. Microscopic observation revealed irregular damage and loss of shape of mycelia upon thymol exposure. Thymol induced the accumulation of superoxide radical in mycelial cells and accompanied increased activity of antioxidative enzymes (SOD, superoxide dismutase; CAT, catalase). Thymol induced membrane permeability was indicated by lipid peroxidation and electrolyte leakage (increased by 29–58%) in mycelial cells. These results suggested that thymol induced oxidative damage in mycelia, which may be one of the possible reasons for thymol-induced mycelial cell death observed with fluorescent detection. Thymol decreased the production of conidia and inhibited the germination of conidia. Thymol induced superoxide radical accumulation, lipid peroxidation, and cell death in conidia as well. All of these results revealed the inhibitory activity of thymol against FON, which may have resulted from the superoxide radical-induced oxidative injury in both conidia and mycelia of FON.

Histochemical and surface microstructural analyses of floral cuticles provide evidence for differential behaviors in scent volatiles emission in two tuberoses (Agave amica (Medik.) Theide and Govaerts) cultivars from Asparagaceae

Two popular cultivars of tuberose (Agave amica (Medik.) Theide and Govaerts syn. Polianthes tuberosa L.) from the family Asparagaceae, widely cultivated for floricultural purposes are ,'Calcutta Single' (CS) and 'Calcutta Double' (CD). It was evidenced that CS flowers emit intense scent volatiles as compared to CD flowers, while CD flowers retained higher contents of internal pool of scent volatiles than CS flowers. Such contrasting observations between the flowers of two cultivars suggest the role of possible barriers such as cuticles that prevent the emission of scent volatiles. The cuticular structure of both cultivars was studied using different histochemical approaches, including light and epi-fluorescence microscopies. Scanning electron microscopy (SEM) was used to understand the cuticular deposition in the cultivars. Our observations confirmed relatively higher cuticular thickness in CD flowers than in CS. SEM results showed the accumulation of wax crystalloids in CS flower and the formation of cuticular nanoridges in CD flowers over the surfaces of the tepals. In addition, accumulation of lipid droplets was also noticed on the tepal peels of the cultivars showing a denser deposition in CD. Our observations suggest that the presence of wax crystalloids in CS flowers enhances the emission of volatiles, while in CD flowers, the presence of thick cuticular nanoridges possibly deters the scent emission and accumulated as an internal pool in higher amounts. The stomatal behavior also indicates a proportional relationship between cuticular deposition and scent emission; low cuticular deposition corresponds to higher emission of floral volatiles emission and vice versa as observed in CS and CD flowers, respectively.

Assessing the interactions between nanoparticles and biological barriers in vitro: a new challenge for microscopy techniques in nanomedicine.

Nanoconstructs intended to be used as biomedical tool must be assessed for their capability to cross biological barriers. However, studying in vivo the permeability of biological barriers to nanoparticles is quite difficult due to the many structural and functional factors involved. Therefore, the in vitro modeling of biological barriers -2D cell monocultures, 2D/3D cell co-cultures, microfluidic devices- is gaining more and more relevance in nanomedical research. Microscopy techniques play a crucial role in these studies, as they allow both visualizing nanoparticles inside the biological barrier and evaluating their impact on the barrier components. This paper provides an overview of the various microscopical approaches used to investigate nanoparticle translocation through in vitro biological barrier models. The high number of scientific articles reported highlights the great contribution of the morphological and histochemical approach to the knowledge of the dynamic interactions between nanoconstructs and the living environment.

Enzyme histochemistry: a useful tool for examining the spatial distribution of brain ectonucleotidases in (patho)physiological conditions.

Adenosine 5'-triphosphate (ATP) and other nucleotides and nucleosides, such as adenosine, are versatile signaling molecules involved in many physiological processes and pathological conditions in the nervous system, especially those with an inflammatory component. They can be released from nerve cells, glial cells, and vascular cells into the extracellular space where they exert their function via ionotropic (P2X) or metabotropic (P2Y) receptors. Signaling via extracellular nucleotides and adenosine is regulated by cell-surface located enzymes ectonucleotidases that hydrolyze the nucleotide to the respective nucleoside. This review summarizes a histochemical approach for detection of ectonucleotidase activities in the cryo-sections of brain tissue. The enzyme histochemistry (EHC) might be used as suitable replacement for immunohistochemistry, since it gives information about both localization and activity, thus adding a functional component to a classical histological approach. With this technique, it is possible to visualize spatial distribution and cell-specific localization of ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) and ecto-5'-nucleotidase (eN/CD73) activities during brain development, after different hormonal manipulations, during neurodegeneration, etc. EHC is also suitable for investigation of microglial morphology in different (patho)physiological conditions. Furthermore, the review describes how to quantify EHC results.

Ovarian morphology and oocyte development in the seabob shrimp Xiphopenaeus kroyeri: a histological and histochemical approach for the study of penaeid reproduction

Histological and histochemical changes during oocyte development were evaluated in order to establish a model for better understanding nutrient dynamics throughout vitellogenesis and overall penaeid reproduction. Samples of Xiphopenaeus kroyeri females (n = 587) at different stages of gonadal maturation were subjected to histological and histochemical analysis for the identification of neutral polysaccharides, total proteins, and tissue differentiation. We observed that the ovarian structure was composed of collagen fibers and oviducts were lined by three layers of epithelium separated by muscular tissue. Oogenesis was classified into six distinct phases: chromatin nucleolar oocytes, chromatin perinucleolar oocytes, oocytes in primary vitellogenesis, oocytes in secondary vitellogenesis, mature oocytes, and atretic oocytes. Histochemical observations suggested that during the previtellogenesis phase, proteins and neutral polysaccharides were not present. However, a progressive increase in these substances occurred from vitellogenic oocytes until their final maturation stage. Follicular cells composed the follicular complex forming an external epithelium in vitellogenic oocytes, and are probably involved in protein transfer during maturation process. Collagen and glycoprotein vesicles were identified in the cytoplasm of mature oocytes of X. kroyeri. Taken together, the present findings contribute to the current knowledge on the ovarian morphology and maturation process of penaeids, supporting future studies on reproductive biology and breeding performance in captivity.

Imaging and spectral analysis of autofluorescence patterns in larval head structures of mosquito vectors.

Autofluorescence (AF) in mosquitoes is currently poorly explored, despite its great potential for investigation of body structures and biological functions. Here, for the first time AF in larval heads of two mosquitoes of key public health importance, Aedes albopictus (Skuse) and Culex pipiens Linnaeus (Diptera: Culicidae), is studied using fluorescence imaging and spectrofluorometry, similar to a label-free histochemical approach. While presenting a generally conserved distribution, AF emission signals show differences in their localization both between mouth brushes and antennae of the two species. The blue AF ascribable to resilin is detected in a more extended area at the antennal bases in Cx. pipiens than in Ae. albopictus, suggesting a potential need to support different antennal movements. The AF spectra, larger in Cx. pipiens than in Ae. albopictus, indicate differences in material composition and molecular properties between the two species likely relatable to their biology, including diverse feeding and locomotion behaviors, with implications for vector control.

P-474 Development of a preclinical screening pipeline to test the efficacy of anti-fibrotic and anti-inflammatory drugs on ovarian aging

Abstract Study question Can either systemic or targeted ovarian delivery of anti-inflammatory (Etanercept) or anti-fibrotic (Pirfenidone) drugs have a therapeutic effect on ovarian aging in mice? Summary answer We have established an in vitro and in vivo pre-clinical pipeline in the mouse to evaluate the effect of drugs on ovarian aging. What is known already The female reproductive system is the first to age in humans, beginning when women reach their mid-thirties. Reproductive aging results in infertility and hormonal perturbations which have general health consequences. Most research on reproductive aging has focused on the age-associated reduction in oocyte quantity and quality. However, our lab discovered that the ovarian microenvironment (stroma) in which the gamete develops changes with age. Specifically, a pro-inflammatory milieu develops with a significant increase in pro-inflammatory cytokine production including interleukin-6. Concurrently, collagen I/III accumulates resulting in fibrosis. This fibrotic and inflammatory milieu likely impacts oocyte quality contributing to reproductive aging. Study design, size, duration Etanercept was selected as an anti-inflammatory drug because it binds and inhibits TNF-a/b. Pirfenidone was selected as anti-fibrotic drug because it targets collagen I/III and TGF-b1. In vitro studies were performed using tissue explant-cultures to evaluate the toxicity of the drugs on the ovary. In vivo studies were based on the delivery of these drugs either systemically or targeted to the ovary using mini-osmotic pumps to evaluate the impact of these drugs on ovarian aging. Participants/materials, setting, methods In vitro studies: Ovaries from 7-month (N = 6) and 14-month (N = 6) old CD1 mice were isolated, dissected into equal pieces, and incubated in Pirfenidone (0.05, 0.5, 5mM), Etanercept (1, 10, 100g/ml), or the drug diluent (controls). After 24h the explants were collected and tissue architecture (H&E), collagen content (Picrosirius red/PSR), and cell death (Caspase-3) was analyzed using histochemical approaches. In vivo studies: Alzet mini-osmotic pumps were implanted subcutaneously and 2-delivery approaches tested: systemic (N = 2) and ovarian-targeted(N = 2). Main results and the role of chance We first evaluated the toxicity of Etanercept and Pirfenidone on the ovary using our in vitro system. H&E staining revealed that the structure of the Etanercept and Pirfenidone-treated ovaries were similar to controls. The collagen content was also not significantly different in all groups (p > 0.05, One-way ANOVA, Tukey’s test). However, 5mM Pirfenidone and 10 and 100g/ml Etanercept treatments resulted in a trend of decreased collagen relative to the control ovaries. Cleaved Caspase-3 fluorescent intensity/area was also similar in all conditions (p > 0.05, One-way ANOVA, Tukey’s test). Therefore, our in vitro studies show that Pirfenidone and Etanercept are not toxic demonstrating the potential use of these drugs in the ovary. Next, we tested the use of these drugs in vivo with osmotic pumps in 7-month old mice, when ovarian fibrosis is first observed. As a proof-of-concept, the pump was filled with Alcian Blue (dye). Pumps were implanted subcutaneously for systemic and targeted delivery. For the targeted delivery, the pump was connected to a catheter which was inserted underneath the bursa. The pumps were well tolerated by the mice. Two weeks after surgery the catheters were still attached to the ovaries and none of the ovaries showed structural abnormalities when analyzed with H&E. Limitations, reasons for caution We have established a preclinical pipeline to screen the potential of drugs to extend reproductive longevity. Studies are ongoing to determine the efficacy of Pirfenidone and Etanercept in delaying fibrosis and inflammation in the ovary to improve reproductive outcomes. Experiments to determine the side effects of these drugs are required. Wider implications of the findings Our in vitro studies show that Pirfenidone and Etanercept are not toxic to the ovary suggesting a therapeutic potential of anti-fibrotic and anti-inflammatory drugs in delaying reproductive aging. Our in vivo preclinical pipeline represents a novel system to evaluate the therapeutic effect of these and other drugs on ovarian aging. Trial registration number N/A

Investigating the Ovarian Microstructure in the Genera Helicolenus and Scorpaena (Teleostei, Sub-Order Scorpaenoidei) with Implications for Ovarian Dynamics and Spawning.

Simple SummaryThe diversity of reproductive mechanism in bony fishes is greater than in any other group of vertebrates. It ranges from oviparous, to several stages of viviparous forms. In this context, scorpaenoid fishes belonging to the families Scorpaenidae and Sebastidae are of particular interest, since they show extremely varied reproductive modes connected with ovarian structures. We describe here the ovarian morphology of five rockfish species showing different reproductive modalities, using histology. Specialized microscopic features were found during gametogenesis, strictly related to the production of gelatinous mass surrounding the eggs, typical of these species. Based on microscopic maturity stages here analyzed, we found that all species shed eggs more than once through the spawning season, and were characterized by continuous oogenesis with multiple oocyte deposition. Further ovarian dynamic observations supported the hypothesis that all species had an indeterminate fecundity.The sub-order Scorpenoidei appears to be particularly interesting due to the presence of intermediate stages between oviparity and viviparity in several species. The present study aims to describe the ovarian morphology, using a histological and histochemical approach, in four ovuliparous species belonging to Scorpaena genus compared with a zygoparous species, H. dactylopterus, focusing also on the assessment of the ovarian dynamics in the populations of such species in Sardinia waters (central–western Mediterranean). Ovarian sections of all species were examined using light microscopy. All species showed a specialized ovary, cystovarian type II-3, strictly related to the production of gelatinous matrices surrounding the eggs. Some microscopic peculiarities in the oogenesis process were found: thin zona pellucida, small and low cortical alveoli, and a specialized ovarian wall during the spawning period. All species analyzed were batch-spawners with an asynchronous ovarian organization. A continuous recruitment of oocytes and the occurrence of de novo vitellogenesis was also observed. During the spawning period, low atresia intensity was detected, while a marked increase in this intensity found in the ovaries at the end of spawning season. Our observations may support an indeterminate fecundity type for these species.

Quantitative evaluation of spermatogenesis by fluorescent histochemistry.

Identifying the types of spermatogenic cells that compose seminiferous tubules, as well as qualitative confirmation of the presence or absence of disorders, has been regarded as crucial in spermatogenesis. Sperm count and fertilizing capacity, both of which depend on the quality as well as quantity of spermatogenesis, are factors critical to fertilization. However, the quantitative assessment of spermatogenesis is not commonly practiced. Spermatogenesis has species-specific stages; when the specific stage in the seminiferous tubules is precisely determined, the types of spermatogenic cells in each stage can be spontaneously identified. Thereafter, a unique marker is used to classify the cells observed in each stage. Quantitative assessment of spermatogenesis has the potential to detect inapparent spermatogenesis disorders or numerically indicate the degree of the disorder. To this end, a histochemical approach using unique markers is indispensable for the quantitative assessment of spermatogenesis. Future developments in techniques to measure cell populations using computer software will further facilitate the establishment of quantitative assessment of spermatogenesis as a standard analysis method that can contribute significantly to advance our understanding of spermatogenesis.