Efeitos do estresse térmico na espermatogênese de camundongos: aspectos celulares e epigenéticos
Data
2022-08-25
Autores
Ferreira, Gláucia Helena 
Orientadores
Lopes, Fabíola Freitas de Paula
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O efeito deletério do estresse térmico na fertilidade animal é um problema multifatorial, afetando tecidos e células do sistema reprodutor em mamíferos. Existem evidências de que, no sistema reprodutor de machos púberes, o estresse térmico compromete a espermatogênese, reduzindo a motilidade, a capacidade fecundante do espermatozoide e a fertilidade do animal. No entanto, pouco se sabe sobre os efeitos imediatos e/ou tardios do estresse térmico em animais pré-púberes. Desta forma, este trabalho visou determinar se o estresse térmico aplicado em camundongos machos pré-púberes exerce efeitos imediatos (período pré-púbere) e tardios (período púbere) sobre os aspectos morfológicos das células testiculares bem como determinar os efeitos tardios do estresse térmico no perfil bioquímico e motilidade espermática e na produção in vivo de embriões. Para tanto, casais da linhagem Suiça foram utilizados para obtenção de ninhadas. No 10º dia de desenvolvimento pós-natal (DPN), as matrizes e os filhotes foram aleatoriamente alojados em câmara climática nas condições controle (21°C durante o dia todo) e estresse térmico (35°C por 12 horas do período de luz e 21°C por 12 h durante o período escuro) até́ o 21º dia DPN. Em seguida, os animais foram mantidos na condição controle até o dia da eutanásia. No primeiro experimento, os camundongos foram avaliados quanto a massa corporal, massa testicular e histologia dos testículos nos momentos imediato (dias 22 a 25 DPN) e tardio (dias 91 a 122 DPN). Para caracterização dos efeitos tardios do estresse térmico foi realizada também a contagem espermática, análise computadorizada de espermatozoides (CASA) e perfil bioquímico espermático (Espectroscopia Raman). Para tanto, os testículos direitos foram fixados em 4% (p/v) paraformaldeído, processados para histologia clássica e submetidos à aquisição e análise de imagem. Os testículos e epidídimos esquerdos foram congelados em nitrogênio líquido e destinados às análises de contagem espermática. Os epidídimos direitos foram repicados em meio HTF (human tubal fluid) ou solução tamponada de fosfato (PBS 0,1M) para avaliação do perfil funcional (CASA) ou bioquímico (Raman), respectivamente. Em um segundo experimento, os machos foram submetidos ao estresse térmico no período pré-púbere como previamente descrito. Ao atingirem a puberdade os animais foram acasalados (2 fêmeas para cada macho, com intervalo de 10 dias) para determinação das taxas de blastocistos e estruturas viáveis. A exposição de camundongos pré-púberes ao estresse térmico exerceu um efeito imediato reduzindo o número de espermatócitos (p=0.0076), espermátides arredondadas (p<0,0001) e alongadas (p=0.0016), e células de Leydig (p=0.0065). Além disso o estresse térmico aumentou a porcentagem de túbulos seminíferos com alterações tubulares caraterísticas de atrofia (p=0,0123), vacúolos (p=0,0300), corpos residuais anormais (p=0.0017), perdas parciais de células germinativas (p=0,4159) e redução do epitélio germinativo íntegro (p<.0001). Houve também aumento no número de vacúolos (p<.0001) e corpos residuais anormais (p<.0001) por túbulo. Quando as mesmas características histológicas testiculares foram avaliadas durante a puberdade, o grupo submetido ao estresse térmico apresentou aumento no número de espermatogônias (p=0.0044), espermátides arredondadas (p<0.0001) e células de Sertoli (p<.0001). Em contraste, houve redução no número de espermatócitos (p<0001) e tendência na redução no número de espermátides alongadas (p=0.0832). Além disso o estresse térmico aumentou a porcentagem de túbulos seminíferos com alterações tubulares caraterísticas de corpos residuais anormais (p=0.0002), perdas parciais de células germinativas (p<.0001) e desorganização do epitélio germinativo (p<.0001) e tendência de desorientação de espermátides (p=0.0652). Houve também aumento no número de vacúolos (p=0.0133) e corpos residuais anormais (p=0.0007). O estresse térmico reduziu a massa da cabeça e corpo do epidídimo e aumentou a contagem espermática relativa nessa porção do epidídimo (p=0.0086). Foi observado o aumento na contagem espermática absoluta no testículo (p=0.0131) e na produção diária de espermatozoides (p=0.0131). A exposição de camundongos ao estresse térmico no período pré-púbere aumentou a motilidade progressiva (p=0.0314) mas reduziu a hiperativação espermática (p=0,0239). Os efeitos imediatos e tardios do estresse térmico em machos compactuaram para a redução da fertilidade, com queda na taxa de blastocistos (p=0.0130) e de estruturas viáveis (p=0.0130) e aumento de estruturas degeneradas (p=0.0078) e oócitos não fecundados (p=0.0459) em relação ao grupo controle. Não foram observados efeitos do do estresse térmico sob o perfil bioquímico do espermatozoide. O estresse térmico em camundongos machos pré-púberes exerceu efeito imediato e tardio, comprometendo a histologia dos túbulos seminíferos, a concentração espermática, e alterou os padrões de motilidade espermática acarretando na redução da fertilidade.
The deleterious effect of heat stress on animal fertility is a multifactorial problem, affecting tissues and cells of the reproductive system in mammals. There is evidence that, in the reproductive system of pubescent males, heat stress compromises spermatogenesis, reducing motility, sperm fertilizing capacity and animal fertility. However, little is known about the immediate and/or late effects of heat stress in prepubertal animals. Thus, this project aimed to determine whether heat stress applied to prepubertal male mice exerts immediate (prepubertal period) and late (pubertal period) effects on the morphological aspects of testicular cells as well as to determine the late effects of heat stress on the biochemical profile and sperm motility and in vivo embryo production. For that, pairs of the Swiss lineage were used to obtain litters. On the 10th day of postnatal development (PND), sows and pups were randomly housed in a climate chamber under control conditions (21°C throughout the day) and heat stress (35°C for 12 hours of light and 21°C for 12 h during the dark period) until the 21st day DPN. Then, the animals were kept in the control condition until the day of euthanasia. In the first experiment, mice were evaluated for body mass, testicular mass and testis histology at the immediate (days 22 to 25 PND) and late (days 91 to 122 PND) times. To characterize the late effects of heat stress, sperm count, computerized sperm analysis (CASA) and sperm biochemical profile (Raman Spectroscopy) were also performed. Therefore, the right testes were fixed in 4% (w/v) paraformaldehyde, processed for classical histology and submitted to image acquisition and analysis. The testes and left epididymis were frozen in liquid nitrogen and destined for sperm count analysis. The right epididymis were subcultured in HTF (human tubal fluid) or phosphate buffered solution (PBS 0.1M) for functional (CASA) or biochemical (Raman) profile evaluation, respectively. In a second experiment, males were subjected to pre-pubertal heat stress as previously described. Upon reaching puberty, the animals were mated (2 females for each male, with an interval of 10 days) to determine the rates of blastocysts and viable structures. Exposure of prepubertal mice to heat stress exerted an immediate effect by reducing the number of spermatocytes (p=0.0076), rounded (p<0.0001) and elongated (p=0.0016) spermatids, and Leydig cells (p=0.0065). ). In addition, heat stress increased the percentage of seminiferous tubules with tubular changes characteristic of atrophy (p=0.0123), vacuoles (p=0.0300), abnormal residual bodies (p=0.0017), partial loss of germ cells (p=0.0017). =0.4159) and reduction of intact germinal 10 epithelium (p<.0001). There was also an increase in the number of vacuoles (p<.0001) and abnormal residual bodies (p<.0001) per tubule. When the same testicular histological characteristics were evaluated during puberty, the group submitted to heat stress showed an increase in the number of spermatogonia (p=0.0044), rounded spermatids (p<0.0001) and Sertoli cells (p<.0001). In contrast, there was a reduction in the number of spermatocytes (p<0001) and a trend towards a reduction in the number of elongated spermatids (p=0.0832). In addition, heat stress increased the percentage of seminiferous tubules with tubular alterations characteristic of abnormal residual bodies (p=0.0002), partial loss of germ cells (p<.0001) and disorganization of the germinal epithelium (p<.0001) and tendency to disorientation of spermatids (p=0.0652). There was also an increase in the number of vacuoles (p=0.0133) and abnormal residual bodies (p=0.0007). Heat stress reduced epididymal head and body mass and increased relative sperm count in this portion of the epididymis (p=0.0086). An increase in the absolute sperm count in the testis (p=0.0131) and in the daily production of spermatozoa (p=0.0131) was observed. Exposure of mice to heat stress in the prepubertal period increased progressive motility (p=0.0314) but reduced sperm hyperactivation (p=0.0239). The immediate and late effects of heat stress in males contributed to the reduction of fertility, with a decrease in the rate of blastocysts (p=0.0130) and of viable structures (p=0.0130) and an increase in degenerated structures (p=0.0078) and oocytes not fertilized (p=0.0459) in relation to the control group. No effects of heat stress on the sperm biochemical profile were observed. Heat stress in prepubertal male mice exerted an immediate and late effect, compromising the histology of the seminiferous tubules, sperm concentration, and altered sperm motility patterns.
The deleterious effect of heat stress on animal fertility is a multifactorial problem, affecting tissues and cells of the reproductive system in mammals. There is evidence that, in the reproductive system of pubescent males, heat stress compromises spermatogenesis, reducing motility, sperm fertilizing capacity and animal fertility. However, little is known about the immediate and/or late effects of heat stress in prepubertal animals. Thus, this project aimed to determine whether heat stress applied to prepubertal male mice exerts immediate (prepubertal period) and late (pubertal period) effects on the morphological aspects of testicular cells as well as to determine the late effects of heat stress on the biochemical profile and sperm motility and in vivo embryo production. For that, pairs of the Swiss lineage were used to obtain litters. On the 10th day of postnatal development (PND), sows and pups were randomly housed in a climate chamber under control conditions (21°C throughout the day) and heat stress (35°C for 12 hours of light and 21°C for 12 h during the dark period) until the 21st day DPN. Then, the animals were kept in the control condition until the day of euthanasia. In the first experiment, mice were evaluated for body mass, testicular mass and testis histology at the immediate (days 22 to 25 PND) and late (days 91 to 122 PND) times. To characterize the late effects of heat stress, sperm count, computerized sperm analysis (CASA) and sperm biochemical profile (Raman Spectroscopy) were also performed. Therefore, the right testes were fixed in 4% (w/v) paraformaldehyde, processed for classical histology and submitted to image acquisition and analysis. The testes and left epididymis were frozen in liquid nitrogen and destined for sperm count analysis. The right epididymis were subcultured in HTF (human tubal fluid) or phosphate buffered solution (PBS 0.1M) for functional (CASA) or biochemical (Raman) profile evaluation, respectively. In a second experiment, males were subjected to pre-pubertal heat stress as previously described. Upon reaching puberty, the animals were mated (2 females for each male, with an interval of 10 days) to determine the rates of blastocysts and viable structures. Exposure of prepubertal mice to heat stress exerted an immediate effect by reducing the number of spermatocytes (p=0.0076), rounded (p<0.0001) and elongated (p=0.0016) spermatids, and Leydig cells (p=0.0065). ). In addition, heat stress increased the percentage of seminiferous tubules with tubular changes characteristic of atrophy (p=0.0123), vacuoles (p=0.0300), abnormal residual bodies (p=0.0017), partial loss of germ cells (p=0.0017). =0.4159) and reduction of intact germinal 10 epithelium (p<.0001). There was also an increase in the number of vacuoles (p<.0001) and abnormal residual bodies (p<.0001) per tubule. When the same testicular histological characteristics were evaluated during puberty, the group submitted to heat stress showed an increase in the number of spermatogonia (p=0.0044), rounded spermatids (p<0.0001) and Sertoli cells (p<.0001). In contrast, there was a reduction in the number of spermatocytes (p<0001) and a trend towards a reduction in the number of elongated spermatids (p=0.0832). In addition, heat stress increased the percentage of seminiferous tubules with tubular alterations characteristic of abnormal residual bodies (p=0.0002), partial loss of germ cells (p<.0001) and disorganization of the germinal epithelium (p<.0001) and tendency to disorientation of spermatids (p=0.0652). There was also an increase in the number of vacuoles (p=0.0133) and abnormal residual bodies (p=0.0007). Heat stress reduced epididymal head and body mass and increased relative sperm count in this portion of the epididymis (p=0.0086). An increase in the absolute sperm count in the testis (p=0.0131) and in the daily production of spermatozoa (p=0.0131) was observed. Exposure of mice to heat stress in the prepubertal period increased progressive motility (p=0.0314) but reduced sperm hyperactivation (p=0.0239). The immediate and late effects of heat stress in males contributed to the reduction of fertility, with a decrease in the rate of blastocysts (p=0.0130) and of viable structures (p=0.0130) and an increase in degenerated structures (p=0.0078) and oocytes not fertilized (p=0.0459) in relation to the control group. No effects of heat stress on the sperm biochemical profile were observed. Heat stress in prepubertal male mice exerted an immediate and late effect, compromising the histology of the seminiferous tubules, sperm concentration, and altered sperm motility patterns.