Publications by authors named "Rafael H Nóbrega"

14 Publications

  • Page 1 of 1

Interaction between thyroid hormones and gonadotropin inhibitory hormone in ex vivo culture of zebrafish testis: An approach to study multifactorial control of spermatogenesis.

Mol Cell Endocrinol 2021 Jul 24;532:111331. Epub 2021 May 24.

Reproductive and Molecular Biology Group, Department of Structural and Functional Biology, Institute of Biosciences, São Paulo State University (UNESP), 18618-970, Botucatu, São Paulo, Brazil. Electronic address:

Reproduction is under multifactorial control of neurohormones, pituitary gonadotropins, as well as of local gonadal signaling systems including sex steroids, growth factors and non-coding RNAs. Among the factors, gonadotropin-inhibitory hormone (Gnih) is a novel RFamide neuropeptide which directly modulates gonadotropin synthesis and release from pituitary, and in the gonads, Gnih mediated inhibitory actions on gonadotropin response of zebrafish spermatogenesis. Thyroid hormones are peripheral hormones which are also known to interact with reproductive axis, in particular, regulating testicular development and function. This study investigated the interaction between Gnih and thyroid hormones in zebrafish spermatogenesis using in vivo and ex vivo approaches. Three experimental groups were established: "control" (non-treated fish), "methimazole" and "methimazole + T4". Fish were exposed to goitrogen methimazole for 3 weeks; T4 (100 μg/L) was added in the water from the second week only in the "reversal treatment" group. After exposure, testes were dissected out and immediately incubated in Leibovitz's L-15 culture medium containing hCG, Gnih or hCG + Gnih for 7 days. Germ cell cysts and haploid cell population were evaluated by histomorphometry and flow cytometry, respectively. Our results showed that hypothyroidism affected germ cell development in basal and gonadotropin-induced spermatogenesis, in particular, meiosis and spermiogenesis. Hypothyroid testes showed lower amount of spermatozoa, and decreased potency of hCG. We also showed that goitrogen treatment nullified the inhibitory actions of Gnih on the gonadotropin-induced spermatogenesis. This study provided evidences that thyroid hormones are important regulatory factors for hCG- and Gnih-mediated functions in zebrafish spermatogenesis.
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http://dx.doi.org/10.1016/j.mce.2021.111331DOI Listing
July 2021

Molecular characterization and expression analysis of anti-Müllerian hormone in common carp (Cyprinus carpio) adult testes.

Gene Expr Patterns 2021 Jun 3;40:119169. Epub 2021 Mar 3.

Reproductive and Molecular Biology Group, Department of Structural and Functional Biology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil. Electronic address:

Anti-Müllerian hormone (Amh) is a member of the transforming growth factor-β (Tgf-β) superfamily required in the regression of Müllerian ducts during gonadal sex differentiation of higher vertebrates. Teleost fish lack Müllerian ducts, but identified Amh orthologs have been shown to exert crucial functions during sex determination and differentiation of several species of teleosts. However, the function of Amh during gametogenesis in adult fish remains poorly investigated. Therefore, to expand present knowledge on the role of Amh in teleosts, the present study aimed to isolate and clone full-length amh cDNA in the common carp, Cyprinus carpio, and examine its expression levels throughout the male reproductive cycle and in response to different hormone treatments of testicular explants. Molecular cloning and characterization showed that the common carp Amh precursor amino acid sequence shared common features to other fish Amh precursors, including a conserved C-terminus (Tgf-β domain) and a double proteolytic cleavage site (R-X-X-R-X-X-R) upstream to the Tgf-β domain. Expression analysis showed amh dimorphic expression in the adult gonads with higher expression in the testes than ovaries. In testes, amh mRNA was detected in Sertoli cells contacting different types of germ cells, although the expression was greatest in Sertoli cells associated with type A undifferentiated spermatogonia. Expression analysis during the reproductive cycle showed that amh transcripts were down-regulated during the developing phase, which is characterized by an increased proliferation of type A undifferentiated spermatogonia and Sertoli cells and appearance of spermatocytes (meiosis) in the testes. Furthermore, ex vivo experiments showed that a 7 day exposure to Fsh or estrogens was required to decrease amh mRNA levels in common carp testicular explants. In summary, this study provided information on the molecular characterization and transcript abundance of amh in common carp adult testes. Altogether, these data will be useful for further investigations on sex determination and differentiation in this species, and also to improved strategies for improved carp aquaculture, such as inhibiting precocious maturation of males.
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http://dx.doi.org/10.1016/j.gep.2021.119169DOI Listing
June 2021

Cystic proliferation of germline stem cells is necessary to reproductive success and normal mating behavior in medaka.

Elife 2021 Mar 1;10. Epub 2021 Mar 1.

Instituto Tecnológico de Chascomús, INTECH (CONICET-UNSAM), Chascomús, Argentina.

The production of an adequate number of gametes is necessary for normal reproduction, for which the regulation of proliferation from early gonadal development to adulthood is key in both sexes. Cystic proliferation of germline stem cells is an especially important step prior to the beginning of meiosis; however, the molecular regulators of this proliferation remain elusive in vertebrates. Here, we report that is an important regulator of cystic proliferation in medaka. We generated mutants of that led to a disruption of cystic proliferation of germ cells. This loss of cystic proliferation was observed from embryogenic to adult stages, impacting the success of gamete production and reproductive parameters such as spawning and fertilization. Interestingly, the depletion of cystic proliferation also impacted male sexual behavior, with a decrease of mating vigor. These data illustrate why it is also necessary to consider gamete production capacity in order to analyze reproductive behavior.
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http://dx.doi.org/10.7554/eLife.62757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7946426PMC
March 2021

RADSex: A computational workflow to study sex determination using restriction site-associated DNA sequencing data.

Mol Ecol Resour 2021 Jul 9;21(5):1715-1731. Epub 2021 Mar 9.

Physiological Chemistry, Biocenter, University of Wuerzburg, Wuerzburg, Germany.

The study of sex determination and sex chromosome organization in nonmodel species has long been technically challenging, but new sequencing methodologies now enable precise and high-throughput identification of sex-specific genomic sequences. In particular, restriction site-associated DNA sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software specifically designed to search for and visualize sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about the type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analysed a published data set of Japanese medaka, Oryzias latipes, where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyse new RAD-Seq data sets from 15 fish species spanning multiple taxonomic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in nonmodel species thanks to its speed of analyses, low resource usage, ease of application and visualization options. Furthermore, our analysis of new data sets from 15 species provides new insights on sex determination in fish.
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http://dx.doi.org/10.1111/1755-0998.13360DOI Listing
July 2021

Effects of gonadotropin-inhibitory hormone on early and late stages of spermatogenesis in ex-vivo culture of zebrafish testis.

Mol Cell Endocrinol 2021 01 27;520:111087. Epub 2020 Nov 27.

Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta, T2N 1N4, Canada. Electronic address:

Gonadotropin-inhibitory hormone (Gnih) is known to play a role in the regulation of reproduction in vertebrates by influencing gonadotropin release and synthesis. While the endocrine actions of Gnih have been identified in several species, its paracrine/autocrine effects in the control of spermatogenesis are less defined. We have used ex vivo culture of zebrafish testis to investigate the role of gonadal zebrafish Gnih (zGnih) in the regulation of the spermatogenic process. We used FACScan cell cycle analysis, morphometric quantifications, BrdU incorporation and caspase-3 activity assays as well as measuring 11-Ketotestosterone (11-KT) level in the culture media. FACScan analysis and morphometric quantification results demonstrated direct action of zGnih on basal and gonadotropin (Lh and Fsh)-induced spermatogenesis. Treatment with zGnih (10 nM) significantly decreased the number of G0/G1 cells after 7-days of culture while no significant changes were found in the proportion area of spermatogonia cell types. Investigation of DNA synthesis using BrdU (5-Bromo-2'-Deoxyuridine) labeling showed that treatment with zGnih (10 nM) significantly decreased proliferative activity of type A spermatogonia, while increased the mitotic activity of type B spermatogonia. We also showed that treatment with zGnih (100 nM) completely eliminated 11-KT release induced by 100 ng/ml Fsh. Treatment with zGnih (10 and 100 nM) also inhibited both hCG and Fsh-induced spermatogenesis. These results, plus our previous findings, demonstrate that zGnih produced locally in the testis is a component of a complex multifactorial system that regulates testicular function in zebrafish.
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http://dx.doi.org/10.1016/j.mce.2020.111087DOI Listing
January 2021

Cortisol Directly Stimulates Spermatogonial Differentiation, Meiosis, and Spermiogenesis in Zebrafish () Testicular Explants.

Biomolecules 2020 03 10;10(3). Epub 2020 Mar 10.

Reproductive and Molecular Biology Group, Department of Morphology, Institute of Biosciences, São Paulo State University, Botucatu, 18618-970 SP, Brazil.

Cortisol is the major endocrine factor mediating the inhibitory effects of stress on vertebrate reproduction. It is well known that cortisol affects reproduction by interacting with the hypothalamic-pituitary-gonads axis, leading to downstream inhibitory and stimulatory effects on gonads. However, the mechanisms are not fully understood. In this study, we provide novel data demonstrating the stimulatory effects of cortisol on spermatogenesis using an ex vivo organ culture system. The results revealed that cortisol treatment did not modulate basal androgen production, but it influenced transcript levels of a selected number of genes involved in the zebrafish testicular function (androgen receptor), (steroidogenic acute regulatory), (17α-hydroxylase/17,20 lyase/17,20 desmolase), (cytochrome P450, family 11, subfamily A, polypeptide 2), (11-beta hydroxysteroid dehydrogenase), (cytochrome P450, family 2, subfamily K, polypeptide 22), (FKBP prolyl isomerase 5), (glucocorticoid receptor alpha), and (glucocorticoid receptor beta) in a short-term culture. We also showed that cortisol stimulates spermatogonial proliferation and differentiation in an androgen independent manner as well as promoting meiosis and spermiogenesis by increasing the number of spermatozoa in the testes. Moreover, we demonstrated that concomitant treatment with RU 486, a potent glucocorticoid receptor (Gr) antagonist, did not affect the cortisol effects on spermatogonial differentiation but blocked the induced effects on meiosis and spermiogenesis. Supporting the Gr-mediated effects, RU 486 nullified the cortisol-induced expression of (synaptonemal complex protein 3), a marker for the meiotic prophase that encodes a component of the synaptonemal complex. This is consistent with in silico analysis that found 10 putative GREs (glucocorticoid response elements) upstream of the zebrafish . Finally, we also showed that mRNA is expressed in Sertoli and Leydig cells, but also in several types of germ cells, including spermatogonia and spermatocytes. Altogether, this evidence indicates that cortisol exerts paracrine roles in the zebrafish testicular function and spermatogenesis, highlighting its effects on spermatogonial differentiation, meiosis, and spermiogenesis.
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http://dx.doi.org/10.3390/biom10030429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175196PMC
March 2020

Role of GnRH Isoforms in Paracrine/Autocrine Control of Zebrafish (Danio rerio) Spermatogenesis.

Endocrinology 2020 Feb;161(2)

Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.

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http://dx.doi.org/10.1210/endocr/bqaa004DOI Listing
February 2020

Paracrine/autocrine control of spermatogenesis by gonadotropin-inhibitory hormone.

Mol Cell Endocrinol 2019 07 29;492:110440. Epub 2019 Apr 29.

Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta, T2N 1N4, Canada. Electronic address:

Control of testicular development is multifactorial and involves a number of hypothalamic, hypophyseal and peripheral hormones. Here, we investigated direct action of zebrafish gonadotropin-inhibitory hormone (zGnih) which is expressed in the testis, on spermatogenesis in zebrafish, in vitro. Treatment with zGnih at the lower doses (10 and 100 nM) inhibited gonadotropin-induced spermatids/spermatozoa (SPD/SPZ) production. However, at the highest dose (1000 nM), zGnih increased basal number of SPD/SPZ and showed paradoxical effect. The effects of zGnih on testosterone and SPD/SPZ production was blocked in the presence of androgen receptor antagonist, flutamide (FLU). A number of transcripts were also measured to better understand zGnih mechanisms of action on zebrafish spermatogenesis. Our results provide strong support for the hypothesis that locally produced zGnih is a component of the complex multifactorial system that regulates testicular development and function in adult zebrafish, in part, by changes in testicular steroidogenesis and regulation of gonadotropin-induced response.
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http://dx.doi.org/10.1016/j.mce.2019.04.020DOI Listing
July 2019

Glycoprotein Hormone Receptor Knockdown Leads to Reduced Reproductive Success in Male .

Front Physiol 2019 19;10:266. Epub 2019 Mar 19.

Department of Biology, York University, Toronto, ON, Canada.

Glycoprotein hormone receptors mediate a diverse range of physiological functions in vertebrate and invertebrate organisms. The heterodimeric glycoprotein hormone GPA2/GPB5 and its receptor LGR1, constitute a recently discovered invertebrate neuroendocrine signaling system that remains to be functionally characterized. We previously reported that LGR1 is expressed in the testes of adult mosquitoes, where its immunoreactivity is particularly regionalized. Here, we show that LGR1 immunoreactivity is associated with the centriole adjunct of spermatids and is observed transiently during spermatogenesis in mosquitoes, where it may act to mediate the regulation of flagellar development. RNA interference to downregulate LGR1 expression was accomplished by feeding mosquito larvae with bacteria that produced LGR1-specific dsRNA, which led to defects in spermatozoa, characterized with shortened flagella. LGR1 knockdown mosquitoes also retained ∼60% less spermatozoa in reproductive organs and demonstrated reduced fertility compared to controls. To date, the endocrine regulation of spermatogenesis in mosquitoes remains an understudied research area. The distribution of LGR1 and detrimental effects of its knockdown on spermatogenesis in indicates that this heterodimeric glycoprotein hormone signaling system contributes significantly to the regulation of male reproductive biology in this important disease-vector.
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http://dx.doi.org/10.3389/fphys.2019.00266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433794PMC
March 2019

Endocrinology of neotropical vertebrates.

Gen Comp Endocrinol 2019 03 15;273:1-2. Epub 2019 Jan 15.

Instituto de Biociências, Universidade Estadual Paulista (UNESP), Botucatu, Brazil. Electronic address:

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http://dx.doi.org/10.1016/j.ygcen.2019.01.008DOI Listing
March 2019

Characterization of vasa homolog in a neotropical catfish, Jundiá (Rhamdia quelen): Molecular cloning and expression analysis during embryonic and larval development.

Gene 2018 May 14;654:116-126. Epub 2018 Feb 14.

Reproductive and Molecular Biology Group, Department of Morphology, Institute of Bioscience of Botucatu, São Paulo State University, Botucatu, São Paulo, Brazil. Electronic address:

We have characterized the full-length vasa cDNA from Jundiá, Rhamdia quelen (Heptapteridae, Siluriformes). vasa encodes a member of the DEAD-box protein family of ATP-dependent RNA helicases. This protein is highly conserved among different organisms and its role is associated with RNA metabolism. In the majority of the investigated species, vasa is restricted to the germ cell lineage and its expression has been used to study germline development in many organisms, including fish. The deduced R. quelen vasa amino acid sequence displayed high similarity with Vasa protein sequences from other organisms, and did not cluster with PL10 or P68 DEAD-box protein subfamilies. We also reported that there is no other isoform for vasa mRNA in R. quelen gonads. Expression analysis by RT-PCR and qPCR showed vasa transcripts exclusively expressed in the germ cells of R. quelen gonads. R. quelen vasa mRNA was maternally inherited, and was detected in the migrating primordial germ cells (PGCs) until 264 h post-fertilization during embryonic and larval development. This work has characterized for the first time the full-length R. quelen vasa cDNA, and describes its expression patterns during R. quelen embryonic and larval development. Our results will contribute to the basic reproductive biology of this native species, and will support studies using vasa as a germ cell marker in different biotechnological studies, such as germ cell transplantation.
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http://dx.doi.org/10.1016/j.gene.2018.02.029DOI Listing
May 2018

Characterization of Gnrh/Gnih elements in the olfacto-retinal system and ovary during zebrafish ovarian maturation.

Mol Cell Endocrinol 2017 07 8;450:1-13. Epub 2017 Apr 8.

Reproductive and Molecular Biology Group, Department of Morphology, Institute of Bioscience of Botucatu, São Paulo State University, Botucatu, São Paulo, Brazil. Electronic address:

Gonadotropin releasing hormone (GnRH) is one of the key players of brain-pituitary-gonad axis, exerting overall control over vertebrate reproduction. In zebrafish, two variants were characterized and named as Gnrh2 and Gnrh3. In this species, Gnrh3, the hypohysiotropic form, is expressed by neurons of the olfactory-retinal system, where it is related with food detection, intra/interspecific recognition, visual acuity and retinal processing modulation. Previous studies have reported the presence of Gnrh receptors in the zebrafish retina, but not yet in the zebrafish olfactory epithelium. The current study analyzed the presence of gnrh2 and gnrh3, their receptors (gnrhr 1,2,3 and 4) and gnih (gonadotropin inhibitory hormone) transcripts, as well as the Gnrh3 protein in the olfactory epithelium (OE), olfactory bulb (OB), retina and ovary during zebrafish ovarian maturation. We found an increase of gnrh receptors transcripts in the OE at the final stages of ovarian maturation. In the OE, Gnrh3 protein was detected in the olfactory receptor neurons cilia and in the olfactory nerve fibers. Interestingly, in the OB, we found an inverse expression pattern between gnih and gnrh3. In the retina, gnrhr4 mRNA was found in the nuclei of amacrine, bipolar, and ganglion cells next to Gnrh3 positive fibers. In the ovary, gnrh3, gnrhr2 and gnrhr4 transcripts were found in perinucleolar oocytes, while gnih in oocytes at the cortical alveolus stage. Our results suggested that Gnrh/Gnih elements are involved in the neuromodulation of the sensorial system particularly at the final stages of maturation, playing also a paracrine role in the ovary.
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http://dx.doi.org/10.1016/j.mce.2017.04.002DOI Listing
July 2017

Studies in zebrafish reveal unusual cellular expression patterns of gonadotropin receptor messenger ribonucleic acids in the testis and unexpected functional differentiation of the gonadotropins.

Endocrinology 2010 May 22;151(5):2349-60. Epub 2010 Mar 22.

Department of Biology, Division of Endocrinology and Metabolism, Utrecht University, Faculty of Science, Utrecht, The Netherlands.

This study aimed to improve, using the zebrafish model, our understanding of the distinct roles of pituitary gonadotropins FSH and LH in regulating testis functions in teleost fish. We report, for the first time in a vertebrate species, that zebrafish Leydig cells as well as Sertoli cells express the mRNAs for both gonadotropin receptors (fshr and lhcgr). Although Leydig cell fshr expression has been reported in other piscine species and may be a common feature of teleost fish, Sertoli cell lhcgr expression has not been reported previously and might be related to the undifferentiated gonochoristic mode of gonadal sex differentiation in zebrafish. Both recombinant zebrafish (rzf) gonadotropins (i.e. rzfLH and rzfFSH) stimulated androgen release in vitro and in vivo, with rzfFSH being significantly more potent than rzfLH. Forskolin-induced adenylate cyclase activation mimicked, whereas the protein kinase A inhibitor H-89 significantly reduced, the gonadotropin-stimulated androgen release. Therefore, we conclude that both FSH receptor and LH/choriogonadotropin receptor signaling are predominantly mediated through the cAMP/protein kinase A pathway to promote steroid production. Despite this similarity, other downstream mechanisms seem to differ. For example, rzfFSH up-regulated the testicular mRNA levels of a number of steroidogenesis-related genes both in vitro and in vivo, whereas rzfLH or human chorionic gonadotropin did not. Although not fully understood at present, these differences could explain the capacity of FSH to support both steroidogenesis and spermatogenesis on a long-term basis, whereas LH-stimulated steroidogenesis might be a more acute process, possibly restricted to periods during which peak steroid levels are required.
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http://dx.doi.org/10.1210/en.2009-1227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2869266PMC
May 2010

Histological and stereological evaluation of zebrafish (Danio rerio) spermatogenesis with an emphasis on spermatogonial generations.

Biol Reprod 2009 Jul 1;81(1):177-87. Epub 2009 Apr 1.

Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Brazil.

The zebrafish has become an important vertebrate model for basic and biomedical research, including the research field of the biology of reproduction. However, very few morphological and stereological data are available regarding zebrafish testis structure and spermatogenesis. In this careful histomorphometric evaluation of the testis, we studied spermatogonial cells using molecular markers, determined the combined duration of meiotic and spermiogenic phases, and examined the formation of the Sertoli cell barrier (tight junctions). We found at least nine spermatogonial generations and propose a morphology-based nomenclature for spermatogonial generations that is compatible with the one used in higher vertebrates. The number of germ cells per cyst increased dramatically (1 to approximately 1360 cells) from undifferentiated spermatogonia type A to early spermatids. The combined duration of meiotic and spermiogenic phases is approximately 6 days, one of the shorter periods among the teleost fish investigated to date. The number of Sertoli cells per cyst increased 9-fold during the maturational cycle of spermatogenic cysts and stabilized in the meiotic phase at a ratio of approximately 100 early spermatids per Sertoli cell (Sertoli cell efficiency). Similarly to mammals, Sertoli cell proliferation ceased in the meiotic phase, coinciding with the formation of tight junctions between Sertoli cells. Hence, the events taking place during puberty in the germinal epithelium of mammals seem to recapitulate the "life history" of each individual spermatogenic cyst in zebrafish.
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http://dx.doi.org/10.1095/biolreprod.109.076299DOI Listing
July 2009