Types Of Spermatogonia Research Articles

Effects of cold seawater pre-treatments on induction of early sexual maturation and sperm production in European eel (Anguilla anguilla)

To induce sexual maturation in captivity, eels rely on hormonal treatments, but this process is costly and time-consuming. As an alternative, different types of conditioning, also referred as pre-treatment, have been assessed to ease hormonal treatment response. Recent studies have shown that migrating eels experience a wide range of temperatures, varying from 12 °C at night to as low as to 8 °C during the day. Therefore, this study evaluates the effects of low-temperature (10 °C) seawater pre-treatments of different durations (2 and 4 weeks) on male eel reproduction. The eye, gonadosomatic and hepatosomatic indexes from control (without thermic seawater pre-treatment) and pre-treated fish were measured. Blood and testis samples were also collected for sex steroid and histology analysis, respectively. Eels pre-treated for 2 weeks demonstrated increased progestin levels, comparing with the control group. Eels pre-treated for 4 weeks showed significantly higher gonadosomatic index and elevated androgens and estradiol levels in comparison with the remaining groups. In eels pre-treated for 2 and 4 weeks, there was an increase in the proportion of spermatogonia type B cells compared to undifferentiated spermatogonia type A, a differentiation process that was not observed in the control group. Cold seawater pre-treatment induced early sexual maturation, including steroid production, which consequently stimulated biometric changes and increased spermatogonia differentiation. Following the pre-treatments, eels started receiving standard hormonal treatment (with recombinant human chorionic gonadotropin at 20 °C). Pre-treated males started to spermiate earlier than the control group. In some treatment weeks, pre-treated individuals registered higher values of sperm density, motility, and kinetic parameters. Moreover, an economic evaluation was carried out relating the investment made in terms of hormone injections with the volume of high-quality sperm obtained from each experimental group. The low temperature pre-treatments demonstrated their economic effectiveness in terms of hormone treatment profitability, increasing the production of high-quality sperm in the European eel. Thus, this in vivo study suggests that cold seawater pre-treatment may increase sensitivity to the hormone applied during standard maturation treatment.

Superoxidase dismutases (SODs) in the European eel: Gene characterization, expression response to temperature combined with hormonal maturation and possible migratory implications

Superoxide dismutases (SODs) are antioxidant enzymes that protect cells from oxidation. Three SODs have been identified in mammals, but there is limited information in teleosts. This study investigates SODs in the European eel and their expression patterns during testis maturation. Phylogenetic and synteny analyses revealed SODs paralogs and their evolution in vertebrates. The eel possesses one SOD1 and two SOD2/3 (a and b), indicating SOD2 and SOD3 duplication in elopomorphs. SODs expression were then evaluated in various male and female tissues. SOD1 is more expressed in females, while SOD2a and SOD2b dominate brain-pituitary-gonad tissues in both sexes. SOD3a showed predominant expression in the ovary and the male livers, whereas SOD3b was found in the pituitary and brain of both sexes. The effects of different maturation protocols (standard hormonal treatment vs. same protocol preceded with cold seawater pre-treatment) on SODs expression during testis maturation were evaluated. Salinity increase at the onset of standard treatment at 20 °C, simulating early migration, upregulated SOD1, SOD2a, and SOD2b, coinciding with spermatogonia type A differentiated cells dominance. Thereafter, SOD2a and SOD3a decreased, while SOD2b increased during hormonal treatment-induced spermatogenesis. Pre-treatment with seawater at 10 °C, mimicking the conditions at the beginning of the seawater migration, downregulated SOD1 but increased SOD3a expression. Finally, the standard hormonal treatment, replicating spawning at higher temperatures, downregulated SOD1 in eels without any pre-treatment while SOD2a expression increased in pre-treated eels. This study revealed tissue-specific, sex-dependent, and maturation-related SOD expression patterns, predicting SODs dynamic expression profiles during their reproductive migration.

PSVII-6 Effect of Summer Months on Testicular Histoarchitecture and Spermatogenesis in Dezhou Donkey: a Histological Study

Abstract This study aims to illustrate testicular histoarchitecture and progression of spermatogenesis in Dezhou donkeys during summer months. For experiment, 8 male Dezhou donkeys having body weight 260 + 20 kg and aged 2 years were analyzed using microscopy and Jonhson’s score. Based on acrosomal system, Dezhou donkey spermatogenesis comprised of XII stages. Various types of spermatogonia: A1, A2, A3, B1 and B2 were observed along with undifferentiated spermatogonia (Aund; Figure 1, 2, 3 and 4). Testicular histoarchitecture during spermatogenesis, seminiferous tubules diameter (ST), ST height (µm), Luminal tubulaR diameter (µm), Number of ST/field, Number of germ cells along with normal Jonhson’s score illustrated that stages of spermatogenesis and testicular histoarchitecture of adult Dezhou donkeys are not affected during summer months (Table 1).

Molecular functions of Mrhl lncRNA in mouse spermatogenesis.

Mrhl lncRNA regulates the Wnt signaling pathway in mouse spermatogonial cells, resulting in the commitment of B-type spermatogonia to meiotic entry through Mrhl lncRNA-mediated regulation of Sox8. Mrhl lncRNA regulates chromatin dynamics of the Sox8 promoter/enhancer interaction through chromatin looping. Proliferation and meiotic division of spermatogonial stem cells are highly regulated biological processes that occur during spermatogenesis. In addition to protein-coding genes, the mammalian genome encodes thousands of lncRNAs which are spatio-temporally expressed and play key role(s) in cellular differentiation and development. Mrhl lncRNA is one such mono-exonic polyadenylated non-coding RNA encoded within the 15th intron of the mouse Phkb gene. Mrhl lncRNA is expressed in mouse testis amongst other tissues. The RNA is nuclear localized and predominantly bound to chromatin in GC-1 spg cells (derived from B-type spermatogonia). It regulates multiple genes belonging to different biological processes including Wnt signaling. Wnt activation of GC-1 spg cells downregulates Mrhl lncRNA expression, in turn activating the expression of meiotic marker genes and downregulating stem cell markers. We have mapped the genomic loci bound by Mrhl lncRNA and identified 37 genes to be regulated by its physical association. Sox8 gene, one among these, is regulated through its interaction at its promoter through RNA:DNA:DNA triplex structure and chromatin looping mediated by the architectural proteins CTCF and YY1. Here, we summarize our major findings on this novel lncRNA starting from its discovery to biological function(s), particularly during meiotic commitment/initiation in mouse spermatogenesis.

Stereological analysis of spermatogenesis in Brycon orbignyanus (Characiformes: Bryconidae)

Piracanjuba, Brycon orbignyanus, is an important rheophilic species for aquaculture studies on genetic diversity, spermatogenesis, semen cryopreservation and taxonomy. Due to anthropic actions its population has been declining, resulting in the loss of natural stocks, thus pushing the species to near extinction. No stereological analysis of spermatogenesis in piracanjuba has been performed. The present study aimed to evaluate spermatogenesis in B. orbignyanus using stereological analysis to quantify the number of germ cells and Sertoli cells, spermatogonial generations, spermatogenesis yield, meiotic index and the percentage of cell death. Testes of five 1-year-old B. orbignyanus specimens were collected, fixed in 2.5% glutaraldehyde and 4% paraformaldehyde and processed for light microscopy. Three types of spermatogonia were identified. Based on the number of B spermatogonia per cyst, it was estimated that spermatogonia underwent approximately nine mitotic divisions before differentiating into primary spermatocytes. Three types of spermatids were identified. In the final spermatid cyst, there were 1204 germ cells, indicating a spermatogenic yield of 58%. An increase in germ cells resulted in a subsequent increase in the number of Sertoli cells, with growth halted at the spermiogenic phase. Thus, stereological analysis of spermatogenesis is essential for better understanding of the reproductive processes in this species.

RETRACTED: Cinnamomum zeylanicum alleviate testicular damage induced by high fat diet in albino rats; histological and ultrastructural studies

Hyperlipidemia has been related to sever health outcome include cardiovascular complication, metabolic disorders and infertility. Moreover, obesity has also been linked to dangerous effects on testicular morphology, spermatogenesis and sperm malformation. Many studies using different herbal medicines exert protective and therapeutic effect on the testes, spermatogenesis and fertility in animals fed high fat diet. Objective: this study aimed to find out the protective effect of cinnamon on testes of albino rat fed high fat diet (HFD). Forty adult male albino rats were selected and equally divided into 4 groups. Group 1: animals of this group were fed standard diet. Group 2: rats were fed standard diet and cinnamon “15% weight by weight, w/w” for 8 weeks. Group 3: animals in this group were fed HFD (2% cholesterol, 15 % sucrose, 15% corn, 15% cocoa butter, starch and 4.7% cellulose) for 8 weeks. Group 4: animals in this group were fed HFD and cinnamon. At the end of 4 weeks half animals were sacrificed and the rest of animals were sacrificed at the end of 8 weeks and blood samples were collected to assay the testosterone level. As well as testes were taken and prepared for both histological and ultrastructure studies. Histological examination of testicular tissue of HFD-fed animals revealed many pathological changes include degenerated seminiferous tubules, distorted germinal layers and interstitial tissue appeared degenerated with intertubular hemorrhage. Ultrastructural observations showed severe degenerated features including both different types of spermatogonia and interstitial tissue. On the other hand, both histological and ultrastructural alterations were substantially but not completely protect in obese animals fed HFD and cinnamon for 4 weeks while advanced degree of improvement tissue appeared after 8 weeks of the same treatment. As well as, significantly increase in the level of testosterone was recorded when compared with HFD-fed animals. The present work concluded that cinnamon dietary uptake may improve testicular damage induced by HFD as it has anti-inflammatory, anti-obesity and antioxidant activities.

Histological, histochemical, immunohistochemical and ultrastructural characterization of the testes of the dove.

Avian testes have been used in the study of germ cell transfer, importantly for understanding the preservation and control of birds. For this purpose, we use light microscopy, electron microscopy and immunohistochemistry to understand the reproductive efficiency of dove testes. The tunica albuginea was thin and septula testes were not observed. The testicular parenchyma was formed mainly of closely packed convoluted seminiferous tubules with little interstitial area. Three types of spermatogonia were distinguished. The primary spermatocyte appeared as the largest spermatogenic cell and was identified at different stages of meiosis. Different morphological stages of the spermatid were categorized. Various cellular associations were described within the seminiferous epithelium. The cytoplasm of Sertoli cells was pale and ill defined due to its close relationship to the germinal epithelium. The spermatid attached to the luminal border of Sertoli cells and germ cells were closely associated. A single layer of myoid cells surrounded the seminiferous tubule. Testicular telocytes of doves were located in the peritubular region and near the blood vessels. Telopods appeared as long cytoplasmic processes arising from the cell body. Leydig cells were distributed singly or in small groups and cords. The intensity of androgen receptor (AR) immunostaining in the testes of the dove was established for the first time and is described in this paper.

ROLE OF THE REACTIVE OXYGEN AND NITROGEN SPECIES IN THE DEVELOPMENT OF FIBROSIS IN THE TESTES OF RATS WITH PROLONGED CENTRAL DEPRIVATION OF TESTOSTERONE SYNTHESIS

Prostate cancer is the second most common diagnosis in oncology and ranks the fifth among the causes of mortality in men around the world. 1,276,106 cases of newly diagnosed prostate cancer were recorded in 2018. Androgens play a large role in the development of prostate cancer. Elimination of androgens and blockade of their synthesis are key pathogenetic steps in the treatment of prostate cancer. Metabolic and morphological changes in the testes under prolonged central deprivation of testosterone synthesis are currently not well understood. The aim of this study was to establish the role of reactive oxygen and nitrogen species in the development of morphological changes in the testes of rats under prolonged central deprivation of testosterone synthesis. Materials and methods. The experiments were conducted on 20 sexually mature male Wistar rats. Animals were divided into 4 groups of 5 animals in each. The first group (control) received a subcutaneous injection of 0.9% sodium chloride for 180 days. The second group received a subcutaneous injection of diphereline (triptoreline) in a dose of 0.3 mg/kg of active agent for 30 days in order to simulate the central deprivation of testosterone synthesis. In the third group, diphereline was administered in a dose of 0.3 mg/kg of the active agent for 90 days. The fourth group received a subcutaneous injection of diphereline in a dose of 0.3 mg/kg of the active agent for 180 days. Small pieces of testes were fixed and enclosed in paraffin blocks; then 4-μm thick sections were made and stained with hematoxylin and eosin. Histological preparations were studied using an optical microscope with a digital microphotographic nozzle Olympus C 3040-ADU with programs adapted for these studies. The total activity of NO synthases (gNOS), the activity of the inducible isoform of NO synthase (iNOS) and constitutive isoforms (cNOS), as well as the production of superoxide anion radical (O2•-) were determined in 10% homogenate of testes. Results. Long-term central deprivation of testosterone synthesis leads to an increase in the interstitial space volume with the development of fibrotic changes in the testes on the 180th day of the experiment. The wall of seminiferous tubules was compacted, swollen, there were a large number of parietal macrophages from the outside compared to previous periods of the experiment. In the structure of some convoluted seminiferous tubules, first there was discompletion and disorientation, and then desquamation of spermatids. The number of spermatogonia type A and B decreased. Hypochromia and pycnosis were noted in the nuclei. The activities of gNOS and iNOS increased until the 90th day and decreased by 180th when compared with 90th day, remaining higher than the activities of the control group. Activity of cNOS was reduced in all experimental groups. O2•- production increased in all experimental groups. Conclusion: an increase in the production of nitric oxide by the inducible isoform of NO synthase leads to destructive changes in the testes of rats with the subsequent development of fibrotic changes on the 180th day of central deprivation of testosterone synthesis by enhancing the production of superoxide anion radical.

IN VITRO ASSESSMENT OF LONG-TERM EFFECTS OF CYTOTOXIC DRUGS ON MALE REPRODUCTIVE FUNCTION

The purpose of the study was a comparative assessment of long-term toxic effects of cytotoxic drugs (anthracycline antibiotics, topoisomerase activity inhibitors, platinum and taxane complex compounds) on male reproductive function. Material and methods. 2.5-month-old male Wistar rats were used in the experiment. The following cytotoxic drugs were administered to animals in the maximum tolerated dose: Vepesid (etoposide, Teva, Israel), Irinotecan (Campto, Rhone-Poulens, Great Britain), Carboplatin (kemokarb, Dabur India Ltd., India), Paclitaxel (mitotax, Dr. Reddy`s, India), Platidiam (Lachema, Czech Republic), Pharmorubicin (Farmitalia, Carlo Erba). The reproductive status was assessed 90 and 180 days after administration of cytotoxic drugs. The fertilizing ability, productivity of spermatogenesis and the viability of a fertilized egg were evaluated. To determine the sensitivity of different types of spermatogonia to the drugs, we studied the dynamics of the number of their cell populations 2, 5, 10, 15, 30, 90 and 180 days after starting the experiment. Results. In long-term follow-up following administration of farmorubicin and paclitaxel to male rats, a complete (pharmacorubicin) or partial (paclitaxel) decrease in the fertilizing ability due to oligospermia was detected. The decrease in spermatogenesis productivity was reversible. In rats treated with topoisomerase activity inhibitors, the fertilizing ability was preserved, however spermatogenic failure was also seen, as judged by the total sperm number. In the long term after the administration of platinum complexes, no decline in the reproductive function was observed, and the spermatogenesis productivity corresponded to the control values. The severity of toxic effects on gonads was determined by the sensitivity of different types of spermatogonia to the action of drugs. Pharmorubicin and paclitaxel exerted a pronounced toxic effect on stem spermatogonial cells. Paclitaxel and carboplatin induced dominant lethal mutations in spermatogonia, but the likelihood of gametes bearing genetic damage incompatible with life decreased over time.

PSXIII-1 Age-related changes in the ratio of different spermatogonia types in the seminiferous tubules of quail testes

Abstract Spermatogonia are early-undifferentiated germ cells, giving rise to mature male generative cells — the spermatozoa. There are two types of spermatogonia – A and B. Of greatest interest is the use of type A spermatogonia, which are the stem cells of the testes. To select the appropriate age for collecting spermatogonia А from quails it is necessary to know the specific features of spermatogenesis. Development dynamics of various spermatogonia types in the quail testicular tubules was studied. Histological studies of the quails testicular tubules at the age of 1, 2, 3, 4, 5 and 6 weeks (n = 30) were carried out. Samples of testis tissue were fixed in Bouin’s fixative. Histological sections were stained with hematoxylin-eosin. Identification of different spermatogonia types was carried out according to their morphology. Type A spermatogonia were additionally identified by immunohistochemistry using SSEA-1 antibodies. The proportion of spermatogonia in the total number of spermatogenic cells in the seminiferous tubules of quails changed with age. The maximum value was reached at the age of 3 weeks and it was 76±6%. On reaching maturity (6 weeks), this indicator decreased to 12 ± 1 %. In the early period of ontogenesis (1–2 weeks), spermatogonia cells were represented mainly by type A spermatogonia. The proportion of these cells from the total number of spermatogonia reached 80 ± 3 %. With increasing age, this indicator decreased, reaching minimum values for achieving maturity (6 weeks) - 16 ± 1 %. The percentage of type B spermatogonia in the seminiferous tubule of quails on the contrary increased with age — from 5 ± 1% at 1 week old to 70 ± 2% at maturity. Thus, the age no later than 2 weeks is the most optimal for the isolation type A spermatogonia of quails. Supported by RFBR (18-29-07079).

230 Cryopreservation for Canis lupus conservation: Evaluating protocols to freeze grey wolf testicular tissue

Because of the severe decline in grey wolf (Canis lupus) abundance, preservation of genetic diversity within the species is essential to prevent inbreeding depression. Cryopreservation is a valuable tool in endangered species conservation, because it allows genetic material to be rescued from rare individuals for future use. The ability to preserve gonadal tissue is particularly useful in seasonal breeders, such as grey wolves, when mature gametes are not available during the non-breeding season. Optimization of species-specific cryopreservation protocols is necessary due to the toxicity of cryoprotectants. Here, we aimed to investigate three protocols to cryopreserve grey wolf testicular tissue, as a model for endangered canid species. Testicular tissue samples from four adult grey wolves were obtained and cryopreserved using slow-freezing (SF) and needle vitrification (nVT) techniques. Slow-frozen samples were exposed to 15% dimethylsulfoxide (DMSO) or 7.5% DMSO + 7.5% ethylene glycol (EG) in minimum essential medium (MEM) at room temperature, placed in a Mr. Frosty Freezing Container (Thermo Fisher Scientific) and cooled to −80°C overnight, before storage in liquid nitrogen. Needle-vitrified samples were equilibrated in a 7.5% DMSO + 7.5% EG solution in MEM for 10min at 4°C, then treated with 15% DMSO + 15% EG + 0.5M sucrose for 10min at 4°C before plunging into liquid nitrogen. Each sample was thawed using serial dilutions of sucrose (1, 0.5, and 0.25) into MEM at 37°C for 5min each, fixed in 4% paraformaldehyde, and embedded in paraffin wax. Five-micron-thick cross-sections of tissue samples were stained using haematoxylin and eosin. Tubule areas and quantity of each cell type (Sertoli cells, spermatogonia, spermatocytes, spermatids, and abnormal cells) were recorded and compared with a fresh control group for each wolf. Cell populations and tubule areas were analysed using a nonparametric Wilcoxon test through JMP statistical software (SAS Institute Inc.), and are presented as means±standard deviations. There was no difference in tubule areas between the treatment groups (fresh=5200±2870, nVT=5655±2370, SF DMSO=3607±814, SF DMSO+EG=3905±1450µm2; P>0.05). Although there was an increase in abnormal cells/tubule area (µm2) for nVT, SF DMSO, and SF DMSO+EG compared with the fresh samples (0.0015±0.0014, 0.0014±0.0013, 0.0016±0.0011, and 0.0007±0.0004, respectively; P>0.05), differences were not statistically significant. Moreover, the number of Sertoli cells, spermatogonia, and spermatocytes were reduced in the cryopreserved groups compared with fresh tissues, but the differences were not statistically different (P>0.05). The data suggest that the three tested protocols preserved testicular structure and cell populations of cryopreserved testicular tissue and could be useful in the genetic preservation of grey wolves.

Spermatogonial Type 3 Deiodinase Regulates Thyroid Hormone Target Genes in Developing Testicular Somatic Cells.

Premature overexposure to thyroid hormone causes profound effects on testis growth, spermatogenesis, and male fertility. We used genetic mouse models of type 3 deiodinase (DIO3) deficiency to determine the genetic programs affected by premature thyroid hormone action and to define the role of DIO3 in regulating thyroid hormone economy in testicular cells. Gene expression profiling in the neonatal testis of DIO3-deficient mice identified 5699 differentially expressed genes. Upregulated and downregulated genes were, respectively, involved according to DAVID analysis with cell differentiation and proliferation. They included anti-Müllerian hormone and genes involved in the formation of the blood-testis barrier, which are specific to Sertoli cells (SCs). They also included steroidogenic genes, which are specific to Leydig cells. Comparison with published data sets of genes enriched in SCs and spermatogonia, and responsive to retinoic acid (RA), identified a subset of genes that were regulated similarly by RA and thyroid hormone. This subset of genes showed an expression bias, as they were downregulated when enriched in spermatogonia and upregulated when enriched in SCs. Furthermore, using a genetic approach, we found that DIO3 is not expressed in SCs, but spermatogonia-specific inactivation of DIO3 led to impaired testis growth, reduced SC number, decreased cell proliferation and, especially during neonatal development, altered gene expression specific to somatic cells. These findings indicate that spermatogonial DIO3 protects testicular cells from untimely thyroid hormone signaling and demonstrate a mechanism of cross-talk between somatic and germ cells in the neonatal testis that involves the regulation of thyroid hormone availability and action.

Reproductive biology in males of the annual killifish Millerichthys robustus (Cyprinodontiformes: Cynolebiidae)

This study explored the male reproductive biology of the annual killifish Millerichthys robustus; the only annual fish distributed in North America. The structure of the testes was described, as well as the spermatogenesis, spermatozoon and reproductive cycle, using histological sections and gonadosomatic index. The testes of M. robustus correspond to the restricted spermatogonial type, with the spermatogenesis process of cystic and longitudinal type. Spermiation and mature spermatozoa were observed in the network of efferent ducts and in the central duct, uninterruptedly from the third week post hatching (WPH) until pool desiccation, during week 27 WPH. No testes were observed in regression or at rest, indicating the continuous active reproductive phase throughout its adult life. The gonadosomatic index showed that the reproductive potential of males increases towards the end of their life cycle prior to drying up of their pool, suggesting that fish prioritize their energy into reproduction in order to maximize the reproductive success up to the end of their life.

Cold seawater induces early sexual developmental stages in the BPG axis of European eel males

BackgroundThe impossibility of closing the life cycle of the European eel (Anguilla anguilla) in captivity troubles the future of this critically endangered species. In addition, the European eel is a highly valued and demanded resource, thus the successful closing of its life cycle would have a substantial economic and ecological impact. With the aim of obtaining the highest gamete quality, the study of the effects of environmental factors, such as temperature, on reproductive performance may prove valuable. This is especially true for the exposure to cold water, which has been reported to improve sexual development in multiple other Actinopterygii species.ResultsEuropean eel males treated with cold seawater (10 °C, T10) for 2 weeks showed an increase in the proliferation and differentiation of spermatogonial cells until the differentiated spermatogonial type A cell stage, and elevated testosterone and 11-ketotestosterone plasma levels. Transcriptomes from the tissues of the brain-pituitary-gonad (BPG) axis of T10 samples revealed a differential gene expression profile compared to the other experimental groups, with clustering in a principal component analysis and in heat maps of all differentially expressed genes. Furthermore, a functional analysis of differentially expressed genes revealed enriched gene ontology terms involved in the regulation of circadian rhythm, histone modification, meiotic nuclear division, and others.ConclusionsCold seawater treatment had a clear effect on the activity of the BPG-axis of European eel males. In particular, our cold seawater treatment induces the synchronization and increased proliferation and differentiation of specific spermatogonial cells. In the transcriptomic results, genes related to thermoception were observed. This thermoception may have caused the observed effects through epigenetic mechanisms, since all analysed tissues further revealed differentially expressed genes involved in histone modification. The presented results support our hypothesis that a low temperature seawater treatment induces an early sexual developmental stage in European eels. This hypothesis is logical given that the average temperature experienced by eels in the early stages of their oceanic reproductive migration is highly similar to that of this cold seawater treatment. Further studies are needed to test whether a cold seawater treatment can improve the response of European eels to artificial hormonal treatment, as the results suggest.

OR10-4 Spermatogonial Type 3 Deiodinase Regulates Thyroid Hormone Action in Developing Testicular Somatic Cells

The influence of thyroid hormone (TH) on male reproduction is not well understood. Male mice lacking the type 3 deiodinase (DIO3), which inactivates TH, exhibit deficits in testis growth, reproductive hormone levels, spermatogenesis and fertility (Martinez et al, Endocrionology 157:1276, 2016). We aimed to (i) determine the testicular gene expression networks underlying these observations, and (ii) define DIO3 role in regulating TH homeostasis in this tissue. Using RNA-sequencing (n=4), we profiled gene expression in the testis of postnatal day 5 Dio3-/-mice and identified 1607 genes differentially expressed (DEGs) by more than 1.5-fold (q<0.05). The altered expression of 23 genes, some of them involved in steroidogenesis and in the formation of the testis-blood barrier, was confirmed by qPCR. DAVID-based gene ontology analysis revealed a profound functional dichotomy between up- and down-regulated genes. Genes involved in cell proliferation, cell cycle, and chromosome and chromatin organization were markedly overrepresented among 637 down-regulated genes (-Log P value range: 20-56), while genes involved in glycoprotein processing and cell membrane functions were overrepresented among 970 up-regulated genes (-Log P value range: 14-42). DEGs whose expression have also been found altered in a published mouse model of SC-specific androgen receptor deficiency showed an significant inverse correlation in their expression changes between the two models. To understand the cellular roles of DIO3 in the developing testis, we genetically inactivated DIO3 in vivoin Sertoli cells (SCs) using a mouse model carrying a floxed Dio3(Dio3f/f) and a Amh-credriver). This resulted in effective Dio3gene recombination in neonatal testis, but intact levels of DIO3 enzymatic activity, suggesting that DIO3 is not appreciably expressed in SCs. In contrast, DIO3 inactivation in spermatogonia (using Dio3f/fmice carrying a Stra8-cretransgene) resulted in >90% inactivation of DIO3 activity in the postnatal testis, demonstrating that testicular DIO3 is predominantly active in spermatogonia. Furthermore,Dio3f/f/Stra8-cremice manifested significant reductions in testis size and SC number at weaning, although these reductions were moderate compared to those observed in Dio3-/-mice. The testes of Dio3f/f/Stra8-creneonates also exhibited marked alterations in the expression of identified TH-regulated genes specific to SC and Leydig cells. Our findings (i) identify new TH-regulated genes in the developing testis that suggest a critical role for TH in arresting cell proliferation and promoting cell differentiation in this tissue, and (ii) indicate that spermatogonial DIO3 influences TH action in the principal cell types in the tetis, defining a novel cross-talk mechanism between germ and somatic cells in the testis that involves the regulation of TH signaling.

Testicular structure and spermatogenesis of the oviparous goodeids Crenichthys baileyi (Gilbert, 1893) and Empetrichthys latos Miller, 1948 (Teleostei, Cyprinodontiformes).

The cyprinodontiform family Goodeidae comprises some 51 species, including subspecies, of freshwater fishes all of which are at risk or are extinct in the wild. It is classified in two allopatric subfamilies: the Goodeinae, endemic to the Mexican Plateau, and the Empetrichthyinae, known only from relict taxa in Nevada and southern California. The 41 species of goodeins are all viviparous and share a set of well-documented reproductive characters. In contrast, the recent species or subspecies of empetrichthyins are all oviparous and relatively poorly known, yet of critical interest in understanding the evolution of livebearing in the family. We previously described ovarian structure and oogenesis in empetrichthyins using archival museum specimens of females and here extend that study to males. Testicular characters of two species of empetrichthyins, Crenichthys baileyi, and Empetrichthys latos, are studied and compared directly with those of one species of viviparous goodeid, Ataeniobius toweri. The testis is a restricted spermatogonial type in both the Empetrichthyinae and the Goodeinae: spermatogonia are found solely at the distal termini of lobules, a diagnostic character of atherinomorph fishes. Morphology of the differentiation of germinal cells during spermatogenesis is similar in both subfamilies. In the oviparous C. baileyi and E. latos spermatozoa are free in the deferent ducts. In contrast, the spermatozoa of viviparous goodeids are organized into numerous bundles called spermatozeugmata, a characteristic of most fishes that practice internal fertilization. Differences between the goodeid subfamilies are interpreted relative to the oviparous versus viviparous modes of reproduction. Archival museum specimens are a reliable source of data on reproductive morphology, including histology, and may be the only specimens available of rare or extinct taxa.

Testicular structure and spermatogenesis of short mackerel, Rastrelliger brachysoma (Bleeker, 1851) in Upper Gulf of Thailand

Testicular structure and spermatogenesis of short mackerel, Rastrelliger brachysoma (Bleeker, 1851) (Teleostei: Scombridae) was first investigated. The testicular parenchyma was a lobular organ, which was classified as an unrestricted spermatogonial type. The classification of spermatogenetic stage could be classified into six stages based on the pattern of chromatin condensation and other characterizations at the light microscopic level. These six stages included the primary and secondary spermatogonium, primary and secondary spermatocyte, spermatid and spermatozoon. The spermatogenesis could also be classified into another four stages based on the nuclear and cytoplasmic characterizations at the ultrastructural level. Spermatogonium was the early germ cell. It underwent a series of mitotic division to reach the primary spermatocyte. Secondary spermatocyte was shown as the heterochromatin surrounding the nuclear membrane, which was rarely seen within seminiferous lobules. Stages during the spermatids differentiation comprised of the early, intermediate and late stages which are under the degree/change of chromatin condensation. Finally, the spermatozoon was revealed as the aquasperm primitive type. It was composed of an oval head without an acrosome, a short mid-piece consisting of two basal bodies (proximal and distal centrioles) and a long flagella tail without lateral fins. The axonemes of classical form with 9+2 microtubules were presented in the flagellum.

Morfología de testículos de la tuvira (Gymnotus spp.) Sometida a la reproducción por diferentes protocolos hormonales

The aim of study was to analyze the testis morphology of males tuviras (Gymnotus spp.) submitted to reproduction by different hormonal inducers. Forty male tuviras (40g ± 5.0) were used approximately in a completely randomized design with four treatments and 10 replicates; each animal was considered a repetition. For hormonal protocol establishment were tested three inducers: crude extract of carp pituitary (EBHC), equine chorionic gonadotropin (ECG), synthetic analogue of gonadotropin-releasing hormone (GnRH-a) and the control group was administered saline solution at 2%. The three hormones and control were applied in two doses (preparatory and final induction). Ten males per treatment were euthanized for macroscopic and microscopic analysis. It was determined volumetric proportion occupied by the components of testicular parenchyma (seminiferous tubules and intertubular tissue) and quantified populations of spermatogonia type A, type B, primary spermatocytes, secondary spermatocytes, spermatids, Sertoli cells (S) and lumen measurement . The results indicated that the hormone GnRH-a showed a higher capacity to promote the final maturation of the testes (P &lt; 0.05), followed by eCG hormone. The EBHC hormone failed to promote the final maturation in males tuviras.

The efficiency of spermatogenesis and the support capacity of Sertoli cells in Characiformes

This stereological analysis of the types of germ cells and the number of Sertoli cells per cyst in Astyanax altiparanae testes during spermatogenesis is the first such report in Characiformes. Testes of 25 male A. altiparanae were examined. Based on the number of spermatogonia B per cyst (469.2 ± 9.92), we estimated that spermatogonia undergo at least nine mitotic divisions before differentiating into primary spermatocytes. There are four spermatogonia types: undifferentiated spermatogonia A*, undifferentiated spermatogonia, differentiated spermatogonia, and type B spermatogonia. The number of Sertoli cells increased gradually from 1.41 ± 0.51 in the single undifferentiated spermatogonium A* to 9.25 ± 0.50 in cysts of spermatocytes in the leptotene/zygotene stage, possibly related to greater complexity of cellular events during the meiotic stage. The number of germ cells rose dramatically from spermatogonia A (1.0 ± 0) to spermatogonia B (469.2 ± 9.92); however, the quantity of spermatocytes inside the cysts in the leptotene/zygotene stage decreased (300.6 ± 6.97) relative to spermatogonia B, representing a loss of approximately 36% of the former number of cells. This was probably the result of apoptosis, which promotes successful development of the remaining cells during sperm production. The support capacity of Sertoli cells increased gradually during spermatogenesis.

Using curcumin to prevent structural impairments of testicles in rats induced by sodium metabisulfite.

Sodium metabisulfite (Na-MBS) is a disinfectant and preservative agent. Some organ including testicle would be in danger in the case of Na-MBS consumption. Curcumin (CUR) is the constituent of turmeric with protective properties. The effect of CUR on testicles in rats exposed to Na-MBS evaluated using stereological methods. Sprague-Dawley rats were divided into eight groups. The rats in groups I to VIII received the following respectively: distilled water, CUR (100 mg/kg/day), low (0.7 mg/kg/day: acceptable daily intake), intermediate (7 mg/ kg/day), and high (70 mg/kg/day) doses of Na-MBS, and low, intermediate, and high doses of Na-MBS plus CUR. After 7 weeks, the testicles were analyzed. The volume of seminiferous tubule, tubular epithelium and tubule length reduced (25-40 %) on average in the rats that received intermediate and high doses of Na-MBS, while the connective tissue volume increased (15-20 %) in both groups (P<0.01). Besides, 19-36 % and 41-57 % of the cells (spermatogonia types A and B, spermatids, Sertoli and Leydig) were lost in the rats that received intermediate and high doses of Na-MBS respectively in comparison to the control groups. Nonetheless, all the above-mentioned alterations ameliorated drastically in the rats that received Na-MBS plus CUR compared to those exposed to Na-MBS without CUR therapy (P<0.01). The acceptable daily intake of Na-MBS for 7 weeks did not affect on testicular parameters. CUR (100 mg/kg/day) could prevent structural impairments of testicles in the rats induced by Na-MBS (7 and 70 mg/kg/day).