Publications by authors named "Nathália de Lima E Martins Lara"

11 Publications

  • Page 1 of 1

The Proliferation of Pre-Pubertal Porcine Spermatogonia in Stirred Suspension Bioreactors Is Partially Mediated by the Wnt/β-Catenin Pathway.

Int J Mol Sci 2021 Dec 17;22(24). Epub 2021 Dec 17.

Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.

Male survivors of childhood cancer are at risk of suffering from infertility in adulthood because of gonadotoxic chemotherapies. For adult men, sperm collection and preservation are routine procedures prior to treatment; however, this is not an option for pre-pubertal children. From young boys, a small biopsy may be taken before chemotherapy, and spermatogonia may be propagated in vitro for future transplantation to restore fertility. A robust system that allows for scalable expansion of spermatogonia within a controlled environment is therefore required. Stirred suspension culture has been applied to different types of stem cells but has so far not been explored for spermatogonia. Here, we report that pre-pubertal porcine spermatogonia proliferate more in bioreactor suspension culture, compared with static culture. Interestingly, oxygen tension provides an avenue to modulate spermatogonia status, with culture under 10% oxygen retaining a more undifferentiated state and reducing proliferation in comparison with the conventional approach of culturing under ambient oxygen levels. Spermatogonia grown in bioreactors upregulate the Wnt/ β-catenin pathway, which, along with enhanced gas and nutrient exchange observed in bioreactor culture, may synergistically account for higher spermatogonia proliferation. Therefore, stirred suspension bioreactors provide novel platforms to culture spermatogonia in a scalable manner and with minimal handling.
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http://dx.doi.org/10.3390/ijms222413549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8708394PMC
December 2021

GATA-1 mutation alters the spermatogonial phase and steroidogenesis in adult mouse testis.

Mol Cell Endocrinol 2022 02 26;542:111519. Epub 2021 Nov 26.

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

GATA-1 is a transcription factor from the GATA family, which features zinc fingers for DNA binding. This protein was initially identified as a crucial regulator of blood cell differentiation, but it is currently known that the Gata-1 gene expression is not limited to this system. Although the testis is also a site of significant GATA-1 expression, its role in testicular cells remains considerably unexplored. In the present study, we evaluated the testicular morphophysiology of adult ΔdblGATA mice with a mutation in the GATA-1 protein. Regarding testicular histology, GATA-1 mutant mice exhibited few changes in the seminiferous tubules, particularly in germ cells. A high proportion of differentiated spermatogonia, an increased number of apoptotic pre-leptotene spermatocytes (Caspase-3-positive), and a high frequency of sperm head defects were observed in ΔdblGATA mice. The main differences were observed in the intertubular compartment, as ΔdblGATA mice showed several morphofunctional changes in Leydig cells. Reduced volume, increased number and down-regulation of steroidogenic enzymes were observed in ΔdblGATA Leydig cells. Moreover, the mutant animal showed lower serum testosterone concentration and high LH levels. These results are consistent with the phenotypic and biometric data of mutant mice, i.e., shorter anogenital index and reduced accessory sexual gland weight. In conclusion, our findings suggest that GATA-1 protein is an important factor for germ cell differentiation as well as for the steroidogenic activity in the testis.
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http://dx.doi.org/10.1016/j.mce.2021.111519DOI Listing
February 2022

Sperm production and seminal analyses in a Neotropical sperm-storing vespertilionid bat yellowish myotis (Myotis levis).

Theriogenology 2021 Oct 15;174:73-84. Epub 2021 Aug 15.

Laboratory of Cellular Biology, Department of Morphology, Federal University of Minas Gerais - UFMG, Belo Horizonte, MG, Brazil. Electronic address:

Yellowish myotis is a Neotropical vespertilionid bat that presents a seasonal reproduction. The sperm is produced in the Mature stage, stored in the Regressed stage and released in the Rest stage (mating period). Aiming to understand, for the first time, the relationship between testis and epididymis physiology in yellowish myotis reproduction, the spermatogenesis length, sperm production, and seminal parameters were herein evaluated. Fifty-one adult male bats were captured in Santuário do Caraça, Minas Gerais, Brazil. The gonads were collected in the Maturing and Mature stages for histomorphometric and immunohistochemical analyses, whereas the epididymis was evaluated in all reproductive stages for seminal studies. Our results demonstrated that the yellowish myotis spermatogenic process is fast, lasting 31.70 ± 0.15 days. Despite the low Sertoli cell efficiency (6.60 ± 1.23), the high numbers of Sertoli cells per testis enable an elevated sperm production in the Mature stage. The sperm concentration, vitality, and motility presented the highest values in the Regressed stage; however, in this period, an increased incidence of sperm morphological defects was detected. In the following period (Rest stage), a drastic reduction of defective sperm was observed, suggesting quality control of sperm before the mating period. Furthermore, the epididymis ability to maintain a long-term sperm-storage was observed in 26.7% of the bats in the Maturing stage. In summary, yellowish myotis presented a fast and high sperm production during the Mature stage. These sperms are stored and selected before mating period.
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http://dx.doi.org/10.1016/j.theriogenology.2021.08.018DOI Listing
October 2021

Atrazine impairs testicular function in BalB/c mice by affecting Leydig cells.

Toxicology 2021 05 22;455:152761. Epub 2021 Mar 22.

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

Several studies have reported the effects of atrazine on the gonads of many experimental models. However, the short-term effects of in vivo exposure to atrazine on the testes of mice are not well clarified. Here we reported that adult BalB/c mice exposed to atrazine (50 mg kg body weight) by gavage for three consecutive days have reduced numbers of 3β-hydroxysteroid dehydrogenase positive Leydig cells (LCs), associated with increased in situ cell death fluorescence and caspase-3 immuno-expression in the testes. Consequently, immunostaining for cell cycle gene regulators showed increased expressions of p45, accompanied with increased expressions of cyclin D2 and E2. Histological observations of the gonads showed reduced number of germ cells in particular areas, sloughed seminiferous epithelium, presence of giant apoptotic cells close to the seminiferous tubule lumen and in the epididymal lumen along with low numbers of Leydig cells in the testicular interstitial areas. Similarly, LCs isolated from the testes of BalB/c mice that were exposed to atrazine (0.5, 25, 50 mg kg body weight) in the same manner as in the first experiment presented dose-dependent increased caspase-3 activity, decreased cell viability, intratesticular and serum testosterone concentrations and LCs testosterone secretion. In summary, atrazine appears to directly decrease the number of testosterone secreting LCs in mice through apoptosis.
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http://dx.doi.org/10.1016/j.tox.2021.152761DOI Listing
May 2021

Metabolic Requirements for Spermatogonial Stem Cell Establishment and Maintenance In Vivo and In Vitro.

Int J Mol Sci 2021 Feb 18;22(4). Epub 2021 Feb 18.

Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.

The spermatogonial stem cell (SSC) is a unique adult stem cell that requires tight physiological regulation during development and adulthood. As the foundation of spermatogenesis, SSCs are a potential tool for the treatment of infertility. Understanding the factors that are necessary for lifelong maintenance of a SSC pool in vivo is essential for successful in vitro expansion and safe downstream clinical usage. This review focused on the current knowledge of prepubertal testicular development and germ cell metabolism in different species, and implications for translational medicine. The significance of metabolism for cell biology, stem cell integrity, and fate decisions is discussed in general and in the context of SSC in vivo maintenance, differentiation, and in vitro expansion.
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http://dx.doi.org/10.3390/ijms22041998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7922219PMC
February 2021

Targeted Gene Editing in Porcine Spermatogonia.

Front Genet 2020 28;11:627673. Epub 2021 Jan 28.

Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, AB, Canada.

To study the pathophysiology of human diseases, develop innovative treatments, and refine approaches for regenerative medicine require appropriate preclinical models. Pigs share physiologic and anatomic characteristics with humans and are genetically more similar to humans than are mice. Genetically modified pigs are essential where rodent models do not mimic the human disease phenotype. The male germline stem cell or spermatogonial stem cell (SSC) is unique; it is the only cell type in an adult male that divides and contributes genes to future generations, making it an ideal target for genetic modification. Here we report that CRISPR/Cas9 ribonucleoprotein (RNP)-mediated gene editing in porcine spermatogonia that include SSCs is significantly more efficient than previously reported editing with TALENs and allows precise gene editing by homology directed repair (HDR). We also established homology-mediated end joining (HMEJ) as a second approach to targeted gene editing to enable introduction of larger transgenes and/or humanizing parts of the pig genome for disease modeling or regenerative medicine. In summary, the approaches established in the current study result in efficient targeted genome editing in porcine germ cells for precise replication of human disease alleles.
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http://dx.doi.org/10.3389/fgene.2020.627673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876475PMC
January 2021

Regulation of Cell Types Within Testicular Organoids.

Endocrinology 2021 04;162(4)

Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada.

Organoids are 3-dimensional (3D) structures grown in vitro that emulate the cytoarchitecture and functions of true organs. Therefore, testicular organoids arise as an important model for research on male reproductive biology. These organoids can be generated from different sources of testicular cells, but most studies to date have used immature primary cells for this purpose. The complexity of the mammalian testicular cytoarchitecture and regulation poses a challenge for working with testicular organoids, because, ideally, these 3D models should mimic the organization observed in vivo. In this review, we explore the characteristics of the most important cell types present in the testicular organoid models reported to date and discuss how different factors influence the regulation of these cells inside the organoids and their outcomes. Factors such as the developmental or maturational stage of the Sertoli cells, for example, influence organoid generation and structure, which affect the use of these 3D models for research. Spermatogonial stem cells have been a focus recently, especially in regard to male fertility preservation. The regulation of the spermatogonial stem cell niche inside testicular organoids is discussed in the present review, as this research area may be positively affected by recent progress in organoid generation and tissue engineering. Therefore, the testicular organoid approach is a very promising model for male reproductive biology research, but more studies and improvements are necessary to achieve its full potential.
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http://dx.doi.org/10.1210/endocr/bqab033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901658PMC
April 2021

Correction to: Comparative testis structure and function in three representative mice strains.

Cell Tissue Res 2021 Feb;383(2):907-910

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

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http://dx.doi.org/10.1007/s00441-020-03366-8DOI Listing
February 2021

The Sertoli cell: what can we learn from different vertebrate models?

Anim Reprod 2020 May 22;16(1):81-92. Epub 2020 May 22.

Laboratory of Cellular Biology, Department of Morphology, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.

Besides having medical applications, comparative studies on reproductive biology are very useful, providing, for instance, essential knowledge for basic, conservation and biotechnological research. In order to maintain the reproductive potential and the survival of all vertebrate species, both sperm and steroid production need to occur inside the testis. From the approximately fifty thousand vertebrate species still alive, very few species are already investigated; however, our knowledge regarding Sertoli cell biology is quite good. In this regard, it is already known that since testis differentiation the Sertoli cells are the somatic cells in charge of supporting and orchestrating germ cells during development and full spermatogenesis in adult animals. In the present review, we highlight key aspects related to Sertoli cell biology in vertebrates and show that this key testis somatic cell presents huge and intrinsic plasticity, particularly when cystic (fish and amphibians) and non-cystic (reptiles, birds and mammals) spermatogenesis is compared. In particular, we briefly discuss the main aspects related to Sertoli cells functions, interactions with germ cells, Sertoli cells proliferation and efficiency, as well as those regarding spermatogonial stem cell niche regulation, which are crucial aspects responsible for the magnitude of sperm production. Most importantly, we show that we could greatly benefit from investigations using different vertebrate experimental models, mainly now that there is a big concern regarding the decline in human sperm counts caused by a multitude of factors.
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http://dx.doi.org/10.21451/1984-3143-AR2018-125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7720927PMC
May 2020

Comparative testis structure and function in three representative mice strains.

Cell Tissue Res 2020 Nov 14;382(2):391-404. Epub 2020 Jul 14.

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

Mice are widely used as experimental models due to several positive characteristics and in particular their suitability for studies involving molecular biology and transgenesis. Despite the large number of mice strains currently available, the literature regarding their basic reproductive biology is still relatively scarce. Herein, we comparatively evaluated several important and correlated parameters related to testis structure and function in sexually mature male mice of inbred (C57BL/6, n = 19; BALB/c, n = 17) and outbred (Swiss, n = 17) strains, frequently utilized in research. Swiss mice presented significant variation for many parameters evaluated, including higher sperm production, mainly when compared to the C57BL/6 strain. However, some key parameters such as the duration of spermatogenesis, the Sertoli cell number per testis, and the spermatogenic efficiency were similar among the different strains. Although presenting significantly higher Leydig cell (LC) proportion and numbers per testis gram and per testis, the anogenital index was smaller in Swiss mice. Estradiol levels were lower in C57BL/6, whereas testosterone levels and 3β-HSD expression were similar among strains. Regarding the LC/macrophages relationship, in comparison to the literature, we reported a much higher contribution of macrophages to the mouse intertubule. Thus, we estimated that there are around 1.6 macrophages per LC in BALB/c mice and this intriguing finding could be relevant to testis function in overall and spermatogonial biology in particular. Taken together, our results highlight the importance of knowing more accurately the testis structure and function in the different mice strains available for research, particularly when a specific testis parameter is being investigated.
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http://dx.doi.org/10.1007/s00441-020-03239-0DOI Listing
November 2020

Postnatal testis development in the collared peccary (Tayassu tajacu), with emphasis on spermatogonial stem cells markers and niche.

Gen Comp Endocrinol 2019 03 12;273:98-107. Epub 2018 May 12.

Laboratory of Cellular Biology, Department of Morphology, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil; National Institute for Amazonian Research, Manaus, AM, Brazil. Electronic address:

Collared peccaries (Tayassu tajacu) present a unique testis cytoarchitecture, where Leydig cells (LC) are mainly located in cords around the seminiferous tubules (ST) lobes. This peculiar arrangement is very useful to better investigate and understand the role of LC in spermatogonial stem cells (SSCs) biology and niche. Recent studies from our laboratory using adult peccaries have shown that the undifferentiated type A spermatogonia (A or SSCs) are preferentially located in ST regions adjacent to the intertubular compartment without LC. Following these studies, our aims were to investigate the collared peccary postnatal testis development, from birth to adulthood, with emphasis on the establishment of LC cytoarchitecture and the SSCs niche. Our findings demonstrated that the unique LC cytoarchitecture is already present in the neonate peccary's testis, indicating that this arrangement is established during fetal development. Based on the most advanced germ cell type present at each time period evaluated, puberty (the first sperm release in the ST lumen) in this species was reached at around one year of age, being preceded by high levels of estradiol and testosterone and the end of Sertoli cell proliferation. Almost all gonocytes and SSCs expressed Nanos1, Nanos2 and GFRA1. The analysis of SSCs preferential location indicated that the establishment of SSCs niche is coincident with the occurrence of puberty. Taken together, our findings reinforced and extended the importance of the collared peccary as an animal model to investigate testis function in mammals, particularly the aspects related to testis organogenesis and the SSCs biology and niche.
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http://dx.doi.org/10.1016/j.ygcen.2018.05.013DOI Listing
March 2019
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