Spermatogenic Stages Research Articles

Ganoderma lucidum Polysaccharide Peptide Alleviates Cyclophosphamide-Induced Male Reproductive Injury by Reducing Oxidative Stress and Apoptosis.

Chemotherapy is an important factor leading to male infertility. It is crucial to discover safe and effective treatments to prevent male reproductive injury caused by chemotherapy. The Ganoderma lucidum polysaccharide peptide (GLPP) has multiple pharmacological activities. The purpose of this study was to determine whether GLPP could protect the male sperm production from chemotherapeutic injury using a mouse model, with testicular damage induced by cyclophosphamide (CP). CP (50 mg/kg/day) was injected intraperitoneally into male ICR mice gavaged with different doses of GLPP at certain spermatogenic stages. The experimental results showed that GLPP alleviated the CP-induced reduction in reproductive organ coefficients and sperm parameters and reduced the morphological damage of testicular tissues in a dose-dependent manner. GLPP significantly improved the reproductive index, sperm-related parameters, sex hormone levels, and histological testis architecture at different spermatogenic stages. Furthermore, GLPP significantly increased superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), Nrf2, and HO-1, and decreased malondialdehyde (MDA) and Keap-1 in the testicular tissue, indicating reduced oxidative stress. In addition, GLPP limited CP-induced apoptosis via a reduction in Bax expression and increase in Bcl-2 expression. This study suggests that GLPP plays a protective role in spermatogenesis by reducing chemotherapeutic injury and might be developed into drug for male patients receiving chemotherapy.

Unravelling spermatogenesis in spotted wolffish: Insights from the ultrastructure of juvenile male testes to the cryopreservation of broodstock sperm

The aim of this study was to deepen our understanding of the reproductive biology of male spotted wolffish (Anarhichas minor) using two different experimental approaches involving juvenile and mature broodstock fish. The first approach consisted of a detailed histological examination of the testes to identify the onset of gonadal maturation and characterise the spermatogenic stages in two- and three-year-old juvenile specimens. Light microscopy analysis revealed clear differences between the age groups. Two-year-old fish displayed well-defined interstitial tissue, Sertoli cells and cysts housing spermatogonia stem cells in which meiosis had not yet begun. In contrast, three-year-old fish exhibited cysts containing spermatocytes, spermatids and abundant spermatozoa, indicating the initiation of the spermatogenic cycle, albeit with asynchronous puberty. Histochemical staining revealed a significant presence of smooth myoid cells in the interstitial tissue of sexually mature fish, while electron microscopy further revealed synaptonemal complexes indicating the onset of meiosis and centriolar structures that gave rise to flagella. The second approach focused on optimising semen freezing and cryopreservation procedures in mature broodstock individuals over the age of 10 years. Seven freezing extenders (KT, TS-2, OP, MT, MH, HBSS, or SR), with seawater (SW) as a control, were assessed along with two cryoprotectants dimethylsulfoxide (DMSO) or methanol to evaluate their impact on pre- and post-thaw semen quality. Results showed that the MT and HBSS extenders were superior in total sperm kinetics at 1:3 dilution, and that DMSO showed optimal results in sperm motility and velocity variants. Moreover, the MT and HBSS groups demonstrated consistent sperm viability after cryopreservation, with values similar to fresh samples. Based on the viability results of the SYBR-green-14/PI assay comparing fresh and cryopreserved sperm using MT and HBSS, the MT extender emerged as the most effective freezing medium for cryopreservation of spotted wolffish broodstock sperm. In conclusion, this study provides a comprehensive understanding of the reproductive dynamics of male spotted wolffish, offering valuable insights for both scientific research and aquaculture management.

Uncovering a multitude of stage-specific splice variants and putative protein isoforms generated along mouse spermatogenesis.

Mammalian testis is a highly complex and heterogeneous tissue. This complexity, which mostly derives from spermatogenic cells, is reflected at the transcriptional level, with the largest number of tissue-specific genes and long noncoding RNAs (lncRNAs) compared to other tissues, and one of the highest rates of alternative splicing. Although it is known that adequate alternative-splicing patterns and stage-specific isoforms are critical for successful spermatogenesis, so far only a very limited number of reports have addressed a detailed study of alternative splicing and isoforms along the different spermatogenic stages. In the present work, using highly purified stage-specific testicular cell populations, we detected 33,002 transcripts expressed throughout mouse spermatogenesis not annotated so far. These include both splice variants of already annotated genes, and of hitherto unannotated genes. Using conservative criteria, we uncovered 13,471 spermatogenic lncRNAs, which reflects the still incomplete annotation of lncRNAs. A distinctive feature of lncRNAs was their lower number of splice variants compared to protein-coding ones, adding to the conclusion that lncRNAs are, in general, less complex than mRNAs. Besides, we identified 2,794 unannotated transcripts with high coding potential (including some arising from yet unannotated genes), many of which encode unnoticed putative testis-specific proteins. Some of the most interesting coding splice variants were chosen, and validated through RT-PCR. Remarkably, the largest number of stage-specific unannotated transcripts are expressed during early meiotic prophase stages, whose study has been scarcely addressed in former transcriptomic analyses. We detected a high number of yet unannotated genes and alternatively spliced transcripts along mouse spermatogenesis, hence showing that the transcriptomic diversity of the testis is considerably higher than previously reported. This is especially prominent for specific, underrepresented stages such as those of early meiotic prophase, and its unveiling may constitute a step towards the understanding of their key events.

Deep Learning-Based Spermatogenic Staging in Tissue Sections of Cynomolgus Macaque Testes.

The indirect assessment of adverse effects on fertility in cynomolgus monkeys requires that tissue sections of the testis be microscopically evaluated with awareness of the stage of spermatogenesis that a particular cross-section of a seminiferous tubule is in. This difficult and subjective task could very much benefit from automation. Using digital whole slide images (WSIs) from tissue sections of testis, we have developed a deep learning model that can annotate the stage of each tubule with high sensitivity, precision, and accuracy. The model was validated on six WSI using a six-stage spermatogenic classification system. Whole slide images contained an average number of 4938 seminiferous tubule cross-sections. On average, 78% of these tubules were staged with 29% in stage I-IV, 12% in stage V-VI, 4% in stage VII, 19% in stage VIII-IX, 18% in stage X-XI, and 17% in stage XII. The deep learning model supports pathologists in conducting a stage-aware evaluation of the testis. It also allows derivation of a stage-frequency map. The diagnostic value of this stage-frequency map is still unclear, as further data on its variability and relevance need to be generated for testes with spermatogenic disturbances.

Regulation of early spermatogenesis in the giant prawn Macrobrachium rosenbergii by a GCL homolog†.

The germ cell-less gene is crucial for gonad development in various organisms. Early interventions in its expression suggested a regulatory role at the mitotic stages of spermatogenesis, and its early knockout resulted in complete sterility in Drosophila. Genomic and transcriptomic data available for the catadromous giant prawn Macrobrachium rosenbergii enabled the identification of a germ cell-less homolog for this species, which we termed MroGCL (mRNA accession number OQ533056). An open reading frame containing 494 amino acids and a typical evolutionarily conserved BTB/POZ domain suggests possible protein-protein interaction functions in keeping with the Drosophila germ cell-less protein. Genomic mapping of MroGCL showed a full length of 120896 bases. Analysis of the temporal expression of MroGCL showed constant expression in early prawn embryonic and larval stages, but a significant increase 10days after metamorphosis when crucial sexual differentiation processes occur in prawns. In adult animals, high expression was detected in the gonads compared to the somatic tissues. RNAi-based knock-down experiments showed that both the silenced and control groups reached advanced spermatogenic stages, but that there was a significant decrease in the yield of spermatozoa in about half of the silenced animals. This finding supports our hypothesis that MroGCL is crucial for mitosis during early stage spermatogenesis. In conclusion, this study contributes to the understanding of crustacean gonad development and provides a stepping stone in the development of environmentally valuable sterile crustacean populations.

Metabolic regulation of proteome stability via N-terminal acetylation controls male germline stem cell differentiation and reproduction

The molecular mechanisms connecting cellular metabolism with differentiation remain poorly understood. Here, we find that metabolic signals contribute to stem cell differentiation and germline homeostasis during Drosophila melanogaster spermatogenesis. We discovered that external citrate, originating outside the gonad, fuels the production of Acetyl-coenzyme A by germline ATP-citrate lyase (dACLY). We show that this pathway is essential during the final spermatogenic stages, where a high Acetyl-coenzyme A level promotes NatB-dependent N-terminal protein acetylation. Using genetic and biochemical experiments, we establish that N-terminal acetylation shields key target proteins, essential for spermatid differentiation, from proteasomal degradation by the ubiquitin ligase dUBR1. Our work uncovers crosstalk between metabolism and proteome stability that is mediated via protein post-translational modification. We propose that this system coordinates the metabolic state of the organism with gamete production. More broadly, modulation of proteome turnover by circulating metabolites may be a conserved regulatory mechanism to control cell functions.

Gonadal and Germ Cells Morphology of Nuchequula gerreoides and Eubleekeria splendens (Leiognathidae) from the Pranburi River Estuary of Thailand

The decorated ponyfish Nuchequula gerreoides and splendid ponyfish Eubleekeria splendens are two abundant leiognathid species in the Pranburi River Estuary, Thailand. However, information about their reproductive activity is still limited, preventing the implementation of effective conservative management. In this study, we investigated the sex ratio and the reproductive characteristics in relation to the size distribution of these polyfish species. Fishes were collected during February to November in 2017. The male to female ratios in N. gerreoides and E. splendens were 1.00 : 5.65 (χ2 = 55.2) and 1.00 : 4.65 (χ2 = 40.0), respectively. These ponyfishes shared similar morphology and histology of the reproductive system. All females were classfied as a juvenile because only undeveloped stage (stage 0) oocytes were found in the ovary. Males below 3.4 cm were all juvenile, but all males above 3.4 cm were mature having spermatogenic cells at spermatogenic stages.

Reproductive diapause influences spermatogenesis and testes’ size in the diplochronous wolf spider Allocosa senex (Lycosidae, Araneae) – A case study using a non-experimental approach

Seasonality considerably impacts on the life of organisms and leads to numerous evolutionary adaptations. Some species face seasonal changes by entering a diapause during different life stages. During adulthood, a diapause in the non-reproductive period can affect male gametogenesis as, for example, it occurs in insects. Spiders are distributed worldwide and show a variety of life cycles. However, data on spiders’ life cycles and seasonal adaptations are limited. Here, we explored the effect of reproductive diapause in a seasonal spider for the first time. We used the South American sand-dwelling spider Allocosa senex as a model as this species is diplochronous, meaning that individuals live two reproductive seasons, with juveniles and adults overwintering in burrows. It has been observed that individuals of this species reduce their metabolism during the non-reproductive season, diminishing prey consumption and locomotion to a minimum. This species is also well-known for exhibiting wandering and courting females and sedentary males. We analyzed spermatogenesis throughout the male's life cycle and described the male's reproductive system and spermiogenesis using light and transmission electron microscopy. We found that spermatogenesis in A. senex is asynchronous and continuous. However, when males face the non-reproductive season, the late spermatogenic stages and spermatozoa decline, causing an interruption but not a total arrest of this process. This seasonality is also reflected in smaller testes’ size in males from the non-reproductive season than in other periods. The mechanisms and constraints are unknown, but they could be related to the metabolic depression during this life cycle period. Since sex-role reversal apparently sets a low-intensity sperm competition scenario compared with other wolf spiders, surviving two reproductive seasons may balance mating opportunities by distributing them between both periods. Thus, the partial interruption of spermatogenesis during diapause could allow new mating encounters during the second reproductive season.

Rhythmicity in testicular melatonin and its correlation with the dynamics of spermatogenic cells in an annual reproductive cycle of Clarias batrachus under natural photo-thermal conditions

Melatonin, the pineal hormone, is synthesized and secreted rhythmically in accordance with various environmental cues especially photo-thermal conditions. The reproductive physiology of seasonal breeders is synchronized with the surroundings by melatonin as a neuroendocrine mediator to acts as an important factor in fish reproduction. However, the data on the participation of melatonin in male reproduction and the putative interaction with the process of spermatogenesis in fish is scarce till date. So, major objectives of the current study are to determine for the first time, the relationship, if any, between seasonal levels of melatonin and testicular development and maturation of the germ cells, and also the involvements of specific meteorological parameters in spermatogenesis under natural photo-thermal conditions. We measured the concentration of circulatory and testicular melatonin; value of gonadosomatic index (GSI), relative percentages of different developing spermatogenic cells, area and perimeter (size and shape) of seminiferous lobules along with the level/duration of rainfall, water temperature and day length in six reproductive phases throughout an annual cycle in adult male catfish (Clarias batrachus). Intra-testicular and serum melatonin concentration showed a similar seasonal pattern with a peak during “functional maturity” phase and trough during “slow spermatogenesis” phase. Correlation as well as regression analyses also supported this positive relationship. Interestingly, intra-testicular melatonin also showed a significant positive correlation with GSI and relative percentage as well as lobular size of mature stages (spermatid and spermatozoa) of germ cells in an annual cycle. Furthermore, meteorological factors exhibited as critical cues to regulate the dynamics (in %) of spermatogenic cells and the level of testicular melatonin throughout the annual gonadal cycle. Our results corroborated by principal component (PC) analysis and showed very clearly that active “functional maturity” state is characterized by GSI, testicular melatonin, relative abundance and lobular size of mature spermatogenic stages as key internal oscillators; and studied environmental variables as the external clues for the regulation of spawning process. Collectively, the present data revealed that there is a relationship between melatonin levels and testicular growth and development of germ cells in Clarias batrachus under natural photo-thermal conditions.

Fine ultrastructural features of germ cells and spermatozoa during spermatogenesis in the European grayling, Thymallus thymallus (Teleostei, Salmoniformes, Salmonidae).

This study aimed to examine the ultrastructure of spermatogenic stages and mature spermatozoa in the European grayling, Thymallus thymallus. The testes were examined microscopically with a transmission electron microscope to find out details of the structure and morphology of the grayling germ cells, spermatozoa and some somatic cells. The grayling testis has a tubular shape, with cysts or clusters of germ cells within seminiferous lobules. The spermatogenic cells, including spermatogonia, spermatocytes, and spermatids, can be found along seminiferous tubules. There are electron-dense bodies in germ cells from the primary spermatogonia to secondary spermatocyte stages. These undergo mitosis to reach the secondary spermatogonia stage, when they form primary and secondary spermatocytes. Spermatids undergo three different stages of differentiation during spermiogenesis, characterized by the level of chromatin condensation, elimination of cytoplasm, and the occurrence of the flagellum. The midpiece of spermatozoa is short and contains spherical or ovoid mitochondria. The sperm flagellum has an axoneme with nine doublets of peripheral microtubules and two central microtubules. The result of this study is valuable to be used as a standard reference for germ cell development, which is of great importance to get a clear insight into the process of grayling breeding practice.

Identification of active spermatogenesis using a multiphoton microscope.

The sperm retrieval rate of microdissection testicular sperm extraction varies from 25% to 60%. Therefore, it is necessary to establish objective selection criteria for identifying seminiferous tubules with spermatozoa. Our aim was to develop a method for identifying spermatogenesis without sectioning testicular tissues. Testicular tissues of 10-week-old normal rats were fixed with 4% paraformaldehyde. Fluorescent labeling of seminiferous tubule nuclei and F-actin was performed, and the specimens were observed without sectioning using a multiphoton microscope. Cryptorchid rats were used as a model lacking elongated spermatids. Multiphoton images were compared with images of normal seminiferous tubules. In addition, seminiferous tubules of 10-week-old normal rats were labeled by testicular interstitial injection of fluorescent probes and observed by a multiphoton microscope without fixation. Terminal deoxynucleotidyl transferase dUTP nick end labeling-stained images of normal and probe-injected testes were compared. In fixed seminiferous tubules, elongated spermatids were identified. In addition, F-actin of apical ectoplasmic specialization was observed around elongated spermatids. Furthermore, spermatogenic stages were identified by an array of nuclei or F-actin. In cryptorchid testes, elongated spermatids and F-actin of the apical ectoplasmic specialization were not observed. In testes injected with fluorescent probes, F-actin of the apical ectoplasmic specialization was observed, and spermatogenic stages were identified without fixation. There was no significant difference in the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells per seminiferous tubule between normal and probe-injected testes. Seminiferous epithelium could be observed without sectioning of tissues by fluorescent probes and a multiphoton microscope. Active spermatogenesis was observed by labeling F-actin with and without fixation. Moreover, the toxicity of fluorescent probes was limited. Our method has a potential for live imaging of testicular tissue.

The molecular evolution of spermatogenesis across mammals

The testis produces gametes through spermatogenesis and evolves rapidly at both the morphological and molecular level in mammals1–6, probably owing to the evolutionary pressure on males to be reproductively successful7. However, the molecular evolution of individual spermatogenic cell types across mammals remains largely uncharacterized. Here we report evolutionary analyses of single-nucleus transcriptome data for testes from 11 species that cover the three main mammalian lineages (eutherians, marsupials and monotremes) and birds (the evolutionary outgroup), and include seven primates. We find that the rapid evolution of the testis was driven by accelerated fixation rates of gene expression changes, amino acid substitutions and new genes in late spermatogenic stages, probably facilitated by reduced pleiotropic constraints, haploid selection and transcriptionally permissive chromatin. We identify temporal expression changes of individual genes across species and conserved expression programs controlling ancestral spermatogenic processes. Genes predominantly expressed in spermatogonia (germ cells fuelling spermatogenesis) and Sertoli (somatic support) cells accumulated on X chromosomes during evolution, presumably owing to male-beneficial selective forces. Further work identified transcriptomal differences between X- and Y-bearing spermatids and uncovered that meiotic sex-chromosome inactivation (MSCI) also occurs in monotremes and hence is common to mammalian sex-chromosome systems. Thus, the mechanism of meiotic silencing of unsynapsed chromatin, which underlies MSCI, is an ancestral mammalian feature. Our study illuminates the molecular evolution of spermatogenesis and associated selective forces, and provides a resource for investigating the biology of the testis across mammals.

Spermatozoa HSP90b expression correlates with ROS generation and altered motility in response to methyl parathion treatment in vitro

Objectives: Widely used organophosphorus pesticides, methyl parathion (MePa), alter the reproductive functions in various animals and humans by induction of oxidative stress on augmented release of reactive oxygen species (ROS). MePa affects semen quality by inducing DNA damage through spermatogenic stages. Several heat shock proteins (HSPs) are expressed in response to environmental stressors particularly the redox-active ones for regulation of protein turnover. Since oxidative stress and sperm motility are implicated in MePa toxicity, studying the expression of HSP90b will unravel the mechanism behind its noxiousness. Material and Methods: Spermatozoa isolated from healthy donors were subjected to various concentrations of MePa (50, 250, 500, and 750 μM) for studying its effect on sperm motility, ROS generation, sperm chromatin integrity, and expression of stress responsive molecular chaperone HSP90b. Results: In vitro exposure of MePa at concentrations ≥500 μM results in a decline in sperm motility and an increased generation of ROS, DNA damage, and HSP90b expression. Conclusion: ROS-mediated modulation of HSP90b expression may affect the structural integrity of client proteins and oxidative injury to membrane lipid, along with DNA integrity resulting in declined sperm motility in response to MePa.

A novel pipeline for computerized mouse spermatogenesis staging.

Differentiating 12 stages of the mouse seminiferous epithelial cycle is vital towards understanding the dynamic spermatogenesis process. However, it is challenging since two adjacent spermatogenic stages are morphologically similar. Distinguishing Stages I-III from Stages IV-V is important for histologists to understand sperm development in wildtype mice and spermatogenic defects in infertile mice. To achieve this, we propose a novel pipeline for computerized spermatogenesis staging (CSS). The CSS pipeline comprises four parts: (i) A seminiferous tubule segmentation model is developed to extract every single tubule; (ii) A multi-scale learning (MSL) model is developed to integrate local and global information of a seminiferous tubule to distinguish Stages I-V from Stages VI-XII; (iii) a multi-task learning (MTL) model is developed to segment the multiple testicular cells for Stages I-V without an exhaustive requirement for manual annotation; (iv) A set of 204D image-derived features is developed to discriminate Stages I-III from Stages IV-V by capturing cell-level and image-level representation. Experimental results suggest that the proposed MSL and MTL models outperform classic single-scale and single-task models when manual annotation is limited. In addition, the proposed image-derived features are discriminative between Stages I-III and Stages IV-V. In conclusion, the CSS pipeline can not only provide histologists with a solution to facilitate quantitative analysis for spermatogenesis stage identification but also help them to uncover novel computerized image-derived biomarkers. https://github.com/jydada/CSS. Supplementary data are available at Bioinformatics online.

CAMP-specific phosphodiesterase 8A and 8B isoforms are differentially expressed in human testis and Leydig cell tumor.

Cyclic adenosine monophosphate/Protein kinase A (cAMP/PKA) signaling pathway is the master regulator of endocrine tissue function. The level, compartmentalization and amplitude of cAMP response are finely regulated by phosphodiesterases (PDEs). PDE8 is responsible of cAMP hydrolysis and its expression has been characterized in all steroidogenic cell types in rodents including adrenal and Leydig cells in rodents however scarce data are currently available in humans. Here we demonstrate that human Leydig cells express both PDE8A and PDE8B isoforms. Interestingly, we found that the expression of PDE8B but not of PDE8A is increased in transformed Leydig cells (Leydig cell tumors-LCTs) compared to non-tumoral cells. Immunofluorescence analyses further reveals that PDE8A is also highly expressed in specific spermatogenic stages. While the protein is not detected in spermatogonia it accumulates nearby the forming acrosome, in the trans-Golgi apparatus of spermatocytes and spermatids and it follows the fate of this organelle in the later stages translocating to the caudal part of the cell. Taken together our findings suggest that 1) a specific pool(s) of cAMP is/are regulated by PDE8A during spermiogenesis pointing out a possible new role of this PDE8 isoform in key events governing the differentiation and maturation of human sperm and 2) PDE8B can be involved in Leydig cell transformation.

Morphologic Features and Deep Learning–Based Analysis of Canine Spermatogenic Stages

In nonclinical toxicity studies, stage-aware evaluation is often expected to assess drug-induced testicular toxicity. Although stage-aware evaluation does not require identification of specific stages, it is important to understand microscopic features of spermatogenic staging. Staging of the spermatogenic cycle in dogs is a challenging and time-consuming process. In this study, we first defined morphologic features for the eight spermatogenic stages in standard histology sections (H&E slides) of dog testes. For image analysis, we defined the key morphologic features of five stages/pooled stage groups (I-II, III-IV, V, VI-VII, and VIII). These criteria were used to develop a deep learning (DL) algorithm for staging of the spermatogenic cycle of control dog testes using whole slide images. In addition, a DL-based nucleus segmentation model was trained to detect and quantify the number of different germ cells, including spermatogonia, spermatocytes, and spermatids. Identification of spermatogenic stages and quantification of germ cell populations were successfully automated by the DL models. Combining these two algorithms provided color-coding visual spermatogenic staging and quantitative information on germ cell populations at specific stages that would facilitate the stage-aware evaluation and detection of changes in germ cell populations in nonclinical toxicity studies.

Cross-talk between down-regulation of steroidogenic genes expression and oxidative and apoptotic biomarkers in testes induced by administration of tramadol and boldenone and their combination in male albino rats.

Objective(s):The testis is the male reproductive gland or gonad having two vital functions: to produce both sperm and androgens, primarily testosterone. The study aimed to investigate the effect of tramadol and boldenone injected alone or in combination for 2 months in rats on testicular function.Materials and Methods:Group 1, normal control; Group 2, tramadol HCl (TRAM) (20 mg/kg bwt.) (IP); Group 3, boldenone undecylenate (BOLD) (5 mg/kg bwt) (i.m); Group 4, combination of TRAM (20 mg/kg bwt.) and BOLD (5 mg/kg); respectively for 2 months. Results:TRAM and BOLD alone and in combination showed deteriorated testicular functions, lowered serum steroid levels (FSH, LH, and testosterone), elevation in oxidative biomarkers (MDA & NO) and reduction in GSH and SOD, down-regulation of StaR and HSD17B3 as well as histopathological testicular assessment using H&E staining revealing massive degenerative changes in the seminiferous epithelium and vacuolar changes of most of the spermatogenic stages in both TRAM and BOLD groups. PAS stain showed an intensive reaction in the interstitial tissue between the tubules in the TRAM group. Masson trichrome stain showed abundant collagen fiber deposits in the tunica albuginea with congested BV in the TRAM group. Conclusion:The study illuminated the hazard of administration of these drugs for a long period as well as the prominent deleterious effects reported on concurrent use of both drugs.

Distribution and changes in the sbGnRH system in Rastrelliger brachysoma males during the breeding season

Rastrelliger brachysoma is a mariculture candidate species, but reproduction in captive fish has been problematic. This report examines the difference in the HPG axis, the neuroendocrine system and the development of reproductive tissues between captive vs. wild male R. brachysoma. The gonadosomatic index (GSI) of sexually mature male wild R. brachysoma was 1.12±0.34 and 1.94±0.26 during the non-breeding and breeding seasons, respectively. Captive R. brachysoma had a GSI of 1.88±0.17. All wild R. brachysoma were in the late spermatogenic stage irrespective of seasons. Immunostaining results showed that sbGnRH-immunoreactive neurons were distributed in three areas of the brain, namely the nucleus periventricularis, nucleus preopticus and nucleus lateralis tuberis. Follicle stimulating hormone and luteinizing hormone immunoreactivities were also observed in the pituitary gland. The levels of brain sbGnRH and GtH mRNA were not significantly different between the non-breeding and breeding seasons, but captive fish displayed (times or percent difference) lower mRNA levels than wild fish. These results suggest that these hormones control the testicular development in R. brachysoma and that the impaired reproduction in captivity may be due to their relative lower expression levels of follicle stimulating hormone and luteinizing hormone genes.

Contributions of Flow Cytometry to the Molecular Study of Spermatogenesis in Mammals.

Mammalian testes are very heterogeneous organs, with a high number of different cell types. Testicular heterogeneity, together with the lack of reliable in vitro culture systems of spermatogenic cells, have been an obstacle for the characterization of the molecular bases of the unique events that take place along the different spermatogenic stages. In this context, flow cytometry has become an invaluable tool for the analysis of testicular heterogeneity, and for the purification of stage-specific spermatogenic cell populations, both for basic research and for clinical applications. In this review, we highlight the importance of flow cytometry for the advances on the knowledge of the molecular groundwork of spermatogenesis in mammals. Moreover, we provide examples of different approaches to the study of spermatogenesis that have benefited from flow cytometry, including the characterization of mutant phenotypes, transcriptomics, epigenetic and genome-wide chromatin studies, and the attempts to establish cell culture systems for research and/or clinical aims such as infertility treatment.

Seminiferous epithelium cycle staging based on the development of the acrosome in ram testis.

Testicular histopathology is considered the most sensitive and reliable method to detect the effects of chemicals on sperm production. To carry out a sensitive examination of testicular histopathology and interpret the changes require knowledge of spermatogenic stages. Spermatogenic staging based on acrosome development during spermiogenesis is conventionally performed in animal species routinely used for research and toxicity testing. In contrast, small ruminants, such as sheep and goats, are rarely used as animal models to evaluate toxicity in male reproductive organs. To the best of our knowledge, a comparable spermatogenic staging system in rams has not yet been fully characterised. Hence, this study aimed to adapt the existing spermatogenic staging based on acrosome development in bull testes to fit the seminiferous epithelium cycle of ram testes. The results show that spermatogenic staging based on acrosome development in bull testes can, with slight modifications, be efficiently used for the staging of ram testes.