Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 14 de 14
Filtrar
1.
Reprod Domest Anim ; 59(7): e14661, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38979950

RESUMO

Spermatogonial stem cells (SSCs) comprise the foundation of spermatogenesis and hence have great potential for fertility preservation of rare or endangered species and the development of transgenic animals and birds. Yet, developing optimal conditions for the isolation, culture, and maintenance of SSCs in vitro remains challenging, especially for chicken. The objectives of this study were to (1) find the optimal age for SSC isolation in Huaixiang chicken, (2) develop efficient protocols for the isolation, (3) enrichment, and (4) culture of isolated SSCs. In the present study, we first compared the efficiency of SSC isolation using 11 different age groups (8-79 days of age) of Huaixiang chicken. We found that the testes of 21-day-old chicken yielded the highest cell viability. Next, we compared two different enzymatic combinations for isolating SSCs and found that 0.125% trypsin and 0.02 g/L EDTA supported the highest number and viability of SSCs. This was followed by investigating optimal conditions for the enrichment of SSCs, where we observed that differential plating had the highest enrichment efficiency compared to the Percoll gradient and magnetic-activated cell sorting methods. Lastly, to find the optimal culture conditions of SSCs, we compared adding different concentrations of foetal bovine serum (FBS; 2%, 5%, 7%, and 10%) and different concentrations of GDNF, bFGF, or LIF (5, 10, 20, or 30 ng/mL). We found that a combination of 2% FBS and individual growth factors, including GDNF (20 ng/mL), bFGF (30 ng/mL), or LIF (5 ng/mL), best supported the proliferation and colony formation of SSCs. In conclusion, SSCs can be optimally isolated through enzymatic digestion from testes of 21-day-old chicken, followed by enrichment using differential plating. Furthermore, adding 2% FBS and optimized concentrations of GFNF, bFGF, or LIF in the culture promotes the proliferation of chicken SSCs.


Assuntos
Células-Tronco Germinativas Adultas , Técnicas de Cultura de Células , Separação Celular , Galinhas , Animais , Masculino , Técnicas de Cultura de Células/veterinária , Separação Celular/métodos , Separação Celular/veterinária , Testículo/citologia , Espermatogônias/citologia , Sobrevivência Celular , Células Cultivadas
2.
Cell Tissue Res ; 388(1): 195-210, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35102441

RESUMO

Gonocytes in the neonatal testis have male germline stem cell properties and as such have important potential applications in fertility preservation and regenerative medicine. Such applications require further studies aimed at increasing gonocyte numbers and evaluating their pluripotency in vitro. The objective of the present study was to test the effects of basic fibroblast growth factor (bFGF), glial cell line-derived neurotrophic factor (GDNF), and leukemia inhibitory factor (LIF) on in vitro propagation, colony formation, and expression of pluripotency markers of neonatal porcine gonocytes. Testis cells from 1-week-old piglets were cultured in basic media (DMEM + 15% FBS), supplemented with various concentrations of bFGF, GDNF, and LIF, either individually or in combinations, in a stepwise experimental design. Gonocytes and/or their colonies were evaluated every 7 days and the gonocyte- (DBA) and pluripotency-specific markers (POU5F1, SSEA-1, E-cadherin, and NANOG) assessed on day 28. Greatest gonocyte numbers and largest colonies were found in media supplemented with 10 ng/mL bFGF and 10 ng/mL bFGF + 100 ng/mL GDNF + 1500 U/mL LIF, respectively. The resultant gonocytes and colonies expressed both germ cell- and pluripotency-specific markers. These results shed light on the growth hormone requirements of porcine gonocytes for in vitro proliferation and colony formation.


Assuntos
Fator 2 de Crescimento de Fibroblastos , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Animais , Proliferação de Células , Células Cultivadas , Suplementos Nutricionais , Fator 2 de Crescimento de Fibroblastos/farmacologia , Células Germinativas , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Fator Inibidor de Leucemia/farmacologia , Masculino , Suínos
3.
Cell Tissue Res ; 380(2): 393-414, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32337615

RESUMO

Spermatogonial stem cells (SSCs) are a rare group of cells in the testis that undergo self-renewal and complex sequences of differentiation to initiate and sustain spermatogenesis, to ensure the continuity of sperm production throughout adulthood. The difficulty of unequivocal identification of SSCs and complexity of replicating their differentiation properties in vitro have prompted the introduction of novel in vivo models such as germ cell transplantation (GCT), testis tissue xenografting (TTX), and testis cell aggregate implantation (TCAI). Owing to these unique animal models, our ability to study and manipulate SSCs has dramatically increased, which complements the availability of other advanced assisted reproductive technologies and various genome editing tools. These animal models can advance our knowledge of SSCs, testis tissue morphogenesis and development, germ-somatic cell interactions, and mechanisms that control spermatogenesis. Equally important, these animal models can have a wide range of experimental and potential clinical applications in fertility preservation of prepubertal cancer patients, and genetic conservation of endangered species. Moreover, these models allow experimentations that are otherwise difficult or impossible to be performed directly in the target species. Examples include proof-of-principle manipulation of germ cells for correction of genetic disorders or investigation of potential toxicants or new drugs on human testis formation or function. The primary focus of this review is to highlight the importance, methodology, current and potential future applications, as well as limitations of using these novel animal models in the study and manipulation of male germline stem cells.


Assuntos
Células-Tronco Germinativas Adultas/fisiologia , Espermatogênese/fisiologia , Animais , Humanos
4.
J Therm Biol ; 81: 20-24, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30975419

RESUMO

This study was done to evaluate the effects of Chinese herbal medicine (CHM) and ginger powder on layers-production performance, serum metabolites and antioxidant status under heat stress condition. Two hundred and fifty Lohmann layers were randomly divided into 5 different, including two controls and three experimental groups (H1, H2, and H3). Control groups were fed the basic diet without supplementation, while, the feed of three experimental groups was supplemented with 3.32 g CHM, 10 g ginger powder, and 10 g ginger powder+ 3.32 g CHM per kg of diet, respectively. Results showed that feed consumption and production rate were decreased in the HC group, while, feed intake and production significantly improved when birds were given supplemented diet. The production rate and feed intake of the H3 group were even significantly higher than the NC group. The birds that received supplemented diet had higher glucose level compared to HC. Triglycerides and serum cholesterol had significantly decreased level in supplemented groups compared to HC. Fungal catalase (CAT) level was significantly improved in H2 and H3 groups. Nitric oxide (NO), glutathione peroxidase (GSH-PX) and Total Protein (T-AOC) level were significantly improved in supplemented groups. Findings showed that ginger powder and CHM could be a viable alternative to the synthetic antibiotic in poultry feed.


Assuntos
Antioxidantes/metabolismo , Galinhas/sangue , Galinhas/metabolismo , Medicamentos de Ervas Chinesas/administração & dosagem , Resposta ao Choque Térmico/efeitos dos fármacos , Oviposição/efeitos dos fármacos , Zingiber officinale , Fenômenos Fisiológicos da Nutrição Animal , Animais , Suplementos Nutricionais , Ingestão de Alimentos , Feminino , Temperatura Alta , Medicina Tradicional Chinesa
5.
J Therm Biol ; 79: 112-119, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30612671

RESUMO

Curcumin is a natural phenolic component of yellow curry spice, exhibits antioxidant and anti-inflammatory properties. In this study we investigated whether curcumin suppresses heat-induced apoptosis in chicken embryonic fibroblast cells (CEF) and the underlying mechanism. CEF cells line was divided into 6 groups (4 repetitions per group) including normal temperature group (NC), high temperature control group (H) and 4 experimental groups (H1(5 µmol/L), H2(10 µmol/L), H3(20 µmol/L) and H4(40 µmol/L)). Control groups were cultured in basic medium without Curcumin, while, the experimental groups were supplemented with 5, 10, 20 and 40 µmol/L, respectively. The experimental groups and H control group were cultured at 43 ℃ (95% air/5% CO2), whereas NC group cells were cultured at 37 °C. After 6, 12 and 24 h of culture, cells were collected for viability, proliferation, apoptosis, antioxidant status and gene expression analysis. Results showed that heat stress trigged the ROS production and induced the apoptosis, leading to decrease the cell viability and proliferation. The enzymatic activities of antioxidants (SOD, CAT, and GPX) were down-regulated. The expression of antioxidant enzyme (CAT, SOD1, SOD2, GSTO1, GSTT1 and GSTA3) and MAPK-Nrf2 pathway genes (Nrf2, Jnk, Erk and P38) were down-regulated under heats stress condition. While, the Curcumin treated groups had decreased ROS and MDA content. Down-regulation of the activity and expression of antioxidant enzyme induced by heat were also reversed by curcumin. Furthermore the up-regulation in expression of Nrf2, Jnk, Erk and P38 in supplemented groups revealed the involvement of MAPK-Nrf2 signaling pathway to alleviate oxidative stress induced by heat stress. This study demonstrates that curcumin has the ability to ease the oxidative damage through activating the MAPK-Nrf2 signaling pathway in CEF cells.


Assuntos
Antioxidantes/farmacologia , Curcumina/farmacologia , Fibroblastos/efeitos dos fármacos , Resposta ao Choque Térmico , Sistema de Sinalização das MAP Quinases , Estresse Oxidativo , Animais , Elementos de Resposta Antioxidante , Células Cultivadas , Embrião de Galinha , Fibroblastos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
6.
J Therm Biol ; 78: 131-139, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30509629

RESUMO

Worldwide, the effect of climatic variations has become a great challenge in poultry production. As global climate is changing, it alters the environmental temperatures, precipitation patterns and atmospheric carbon dioxide. Poultry farming mainly depends on climatic conditions such as temperature and humidity. Several factors can be involved but heat stress is one of most important environmental factor influencing a wide range of chickens performances including reduced feed intake which, in turn, affects growth rate, body weight, meat quality, egg quality, egg production, semen quality and fertility; these negative influences result in great economic losses. Heat stress associated food safety issues have gained special importance due to public awareness and an abundance of available scientific information. Environmental modifications (early heat conditioning, open sheds and cooling systems) and nutritional strategies (early feed restriction, electrolyte, vitamin and mineral balance) cannot satisfy the special needs of stressed poultry. Therefore, there exists a crucial need to explore effective strategies including genetic markers to enhance thermo-tolerance and productivity of poultry birds in hot regions of the world.


Assuntos
Criação de Animais Domésticos/métodos , Resposta ao Choque Térmico , Doenças das Aves Domésticas/etiologia , Aves Domésticas/fisiologia , Criação de Animais Domésticos/normas , Animais , Doenças das Aves Domésticas/prevenção & controle , Produtos Avícolas/normas
7.
Reprod Toxicol ; : 108645, 2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38897308

RESUMO

Male reproductive capacity has fallen considerably in recent decades; in addition, the incidence of testicular cancer has increased in many developed countries. The cause of this phenomenon is unknown, but environmental toxicants are considered a major contributing factor. To study potential reproductive toxicants, robust in vitro testis models are needed. We have recently established a porcine testis organoid system with a high resemblance to the architectures of innate testis tissue. Here, we further investigated the testis morphogenesis, cell maturation, and endocrine function of the testis organoids. We also challenged this system with abiraterone, a steroidogenic inhibitor, to validate its suitability as an in vitro platform for endocrine toxicology tests. Our results showed that the testis cells in the organoids reorganize into testis cordal structures, and the cordal relative areas increase in the organoids over time of culture. Moreover, the diameters and cell numbers per cross-section of the cordal structures increased over time. Interestingly, Sertoli cells in the organoids gradually underwent maturational changes by showing increased expression of androgen receptors, decreased expression of the anti-müllerian hormone, and formation of the blood-testis barrier. Next, we confirmed that the organoids respond to hormonal stimulation and release multiple sex hormones, including testosterone, estradiol, and progesterone. Finally, we showed that the production of testosterone and estradiol in this system can be inhibited in response to the steroidogenic inhibitor. Taken together, our organoid system provides a promising in vitro platform for male reproductive toxicology studies on testis morphogenesis, somatic cell maturation, and endocrine production.

8.
Cells ; 12(18)2023 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-37759457

RESUMO

In vitro spermatogenesis (IVS) has important applications including fertility preservation of prepubertal cancer patients; however, thus far, IVS has only been achieved using mouse models. To study the effects of growth factors on the maintenance of testicular tissue integrity, germ cell numbers, and potential induction of IVS using a porcine model, we cultured small testicular fragments (~2 mg) from 1-wk-old piglets under six different media conditions (DMEM + 10%KSR alone or supplemented with GDNF, bFGF, SCF, EGF, or a combination of all) for 8 weeks. Overall, tissues supplemented with GDNF and bFGF had the greatest seminiferous tubule integrity and least number of apoptotic cells. GDNF-supplemented tissues had the greatest number of gonocytes per tubule, followed by bFGF-supplemented tissues. There was evidence of gradual Sertoli cell maturation in all groups. Moreover, histological examination and the expression of c-KIT (a marker of differentiating spermatogonia and spermatocytes) and STRA8 (a marker of the pre/meiotic stage germ cells) confirmed the induction of IVS in all groups. However, GDNF- and bFGF-supplemented tissue cultures had greater numbers of seminiferous tubules with spermatocytes compared to other groups. In conclusion, overall, GDNF and bFGF supplementation better maintained the tissue integrity and gonocyte numbers and induced IVS in cultured testicular tissues.

9.
Animals (Basel) ; 13(1)2022 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-36611737

RESUMO

Long-term culture of testicular tissue has important applications, including the preservation of fertility potential of prepubertal boys undergoing gonadotoxic cancer treatment. This study was designed to define optimal conditions for the long-term culture of neonatal porcine testicular tissue as an animal model for preadolescent individuals. Testes from 1 wk old donor piglets were used to examine the effects of tissue fragment size (~2, 4, 6, or 8 mg), preparation method (intact, semi-digested, or physically dispersed fragments), and serum source in the media (fetal bovine serum­FBS­or knockout serum replacement­KSR). Testicular fragments were examined weekly for 4 weeks for tissue integrity, seminiferous cord density and morphology, and gonocyte counts. Testicular tissue integrity was dependent on fragment size and preparation method, where the smallest size (2 mg, p < 0.05) and intact preparation method were advantageous (p < 0.05). Seminiferous cord density decreased over the culture period (p < 0.05). Although the relative number of gonocytes decreased over time for all sizes and methods (p < 0.01), smaller intact fragments (2 and 4 mg) had greater numbers of gonocytes (p < 0.05). Our findings suggest that intact or physically dispersed testicular fragments of the smallest size (2 mg) cultured in KSR-supplemented media could be effectively maintained in vitro for the duration of 4 weeks.

10.
Cells ; 10(7)2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34359871

RESUMO

The creation of a testis organoid (artificial testis tissue) with sufficient resemblance to the complex form and function of the innate testis remains challenging, especially using non-rodent donor cells. Here, we report the generation of an organoid culture system with striking biomimicry of the native immature testis tissue, including vasculature. Using piglet testis cells as starting material, we optimized conditions for the formation of cell spheroids, followed by long-term culture in an air-liquid interface system. Both fresh and frozen-thawed cells were fully capable of self-reassembly into stable testis organoids consisting of tubular and interstitial compartments, with all major cell types and structural details expected in normal testis tissue. Surprisingly, our organoids also developed vascular structures; a phenomenon that has not been reported in any other culture system. In addition, germ cells do not decline over time, and Leydig cells release testosterone, hence providing a robust, tunable system for diverse basic and applied applications.


Assuntos
Materiais Biomiméticos/farmacologia , Organoides/fisiologia , Testículo/irrigação sanguínea , Animais , Contagem de Células , Criopreservação , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/efeitos dos fármacos , Hormônio Luteinizante/metabolismo , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Especificidade de Órgãos , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/ultraestrutura , Suínos , Testículo/citologia , Testículo/ultraestrutura , Testosterona/metabolismo
11.
Theriogenology ; 162: 84-94, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33450717

RESUMO

The in vitro propagation and differentiation of spermatogonial stem cells (SSCs) has many potential applications within reproductive science and medicine. We established a two-dimensional (2D) cell culture system to proliferate and differentiate prepubertal mouse SSCs as a model capable of maximizing on a small number of donor SSCs. We also investigated the effects of retinol on in vitro SSC differentiation. Testis cells were cultured for 10 days in a serum-free medium. This produced SSC colonies which were then dissociated and sub-cultured for an additional 20 days in a differentiation medium. Before inducing differentiation, colonies expressed genes specific for undifferentiated spermatogonia (Ngn3, Plzf). After 10 days in the differentiation medium, Stra8 expression was upregulated. After 20 days, Acr expression was upregulated, indicating the completion of meiosis. Immunofluorescence, RT-PCR and flow cytometry confirmed the presence of haploid male germ cells (4.4% of all cells). When retinol was added to the differentiation medium the proportion of haploid germ cells increased (8.1% of cells). We concluded that, under serum-free culture conditions, prepubertal SSCs will generate colonies that can differentiate into haploid germ cells in a 2D culture system. These cells demonstrate a relatively high efficiency of haploid-cell production, which can be further improved with retinol.


Assuntos
Células-Tronco Germinativas Adultas , Espermatogônias , Animais , Técnicas de Cultura de Células/veterinária , Diferenciação Celular , Células Cultivadas , Haploidia , Masculino , Camundongos , Espermatogênese , Testículo
12.
Cells ; 9(3)2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32197440

RESUMO

Spermatogonial stem cells (SSCs) are the only adult stem cells capable of passing genes onto the next generation. SSCs also have the potential to provide important knowledge about stem cells in general and to offer critical in vitro and in vivo applications in assisted reproductive technologies. After century-long research, proof-of-principle culture systems have been introduced to support the in vitro differentiation of SSCs from rodent models into haploid male germ cells. Despite recent progress in organotypic testicular tissue culture and two-dimensional or three-dimensional cell culture systems, to achieve complete in vitro spermatogenesis (IVS) using non-rodent species remains challenging. Successful in vitro production of human haploid male germ cells will foster hopes of preserving the fertility potential of prepubertal cancer patients who frequently face infertility due to the gonadotoxic side-effects of cancer treatment. Moreover, the development of optimal systems for IVS would allow designing experiments that are otherwise difficult or impossible to be performed directly in vivo, such as genetic manipulation of germ cells or correction of genetic disorders. This review outlines the recent progress in the use of SSCs for IVS and potential in vivo applications for the restoration of fertility.


Assuntos
Fertilidade , Espermatogênese , Espermatogônias/citologia , Células-Tronco/citologia , Animais , Biomarcadores/metabolismo , Humanos , Masculino , Modelos Biológicos
13.
Biol Futur ; 70(1): 79-87, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34554427

RESUMO

INTRODUCTION: The ability for isolation and in vitro propagation of spermatogonial stem cells (SSCs) offer a base for studies on spermatogenesis, and also contribute to the development of new methods for the preservation of livestock and animal genetic modification. The aim of this study was to find the optimal isolation and culture condition for efficient propagation of SSCs. METHODS: Three different isolation methods (mechanical, one-, and two-step enzymatic digestion) were compared to find the optimal isolation method. To find the best culture conditions for in vitro propagation, isolated SSCs were cultured for 7 days in three different culture conditions supplemented with 10% FBS, 0.25% BSA, and 10% KSR, respectively. RESULTS: The result showed that two-step enzymatic digestion produced a significant high fraction of live cells compared the other two. Non-adhering cells collected after 48 hr and cultured in BSA- and KSR-supplemented medium had a significantly high number of SSCs clump formation compared to FBS-supplemented group. The expression of CD9 confirmed that cell clumps were SSCs clumps. Spermatogonial stem cells cultured in BSA-supplemented medium were positive for NGN3 and PLZF expressions, whereas negative for Stra8 (a meiotic-specific gene) expression, suggesting that most of the cells were undifferentiated SSCs in BSA culture system. In contrast, in FBS- and KSR-supplemented groups, the SSCs were positive forNGN3, PLZF, and Stra8. CONCLUSION: These data revealed that two-step enzymatic digestion is the best method for the isolation, and 0.25% BSA-supplemented culture condition is effective for optimal in vitro propagation of SSCs.

14.
Oncotarget ; 8(39): 66709-66727, 2017 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-29029549

RESUMO

Infertility has become a major health issue in the world. It affects the social life of couples and of all infertility cases; approximately 40-50% is due to "male factor" infertility. Male infertility could be due to genetic factors, environment or due to gonadotoxic treatment. Developments in reproductive biotechnology have made it possible to rescue fertility and uphold biological fatherhood. In vitro production of haploid male germ cell is a powerful tool, not only for the treatment of infertility including oligozoospermic or azoospermic patient, but also for the fertility preservation in pre-pubertal boys whose gonadal function is threatened by gonadotoxic therapies. Genomic editing of in-vitro cultured germ cells could also potentially cure flaws in spermatogenesis due to genomic mutation. Furthermore, this ex-vivo maturation technique with genomic editing may be used to prevent paternal transmission of genomic diseases. Here, we summarize the historical progress of in vitro spermatogenesis research by using organ and cell culture techniques and the future clinical application of in vitro spermatogenesis.

SELEÇÃO DE REFERÊNCIAS
Detalhe da pesquisa