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Comparative genomic identification and expression profiling of CatSper genes in the reproductive tract of the bull
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Abstracts / Animal Reproduction Science 169 (2016) 99–135
P50
P51
Interspecific divergence of sperm morphometry in canids
Comparative genomic identification and expression profiling of CatSper genes in the reproductive tract of the bull
C.S. Asa 1,∗ , L. Vansandt 2 , K. Bauman 1 1
Reproductive and Behavioral Sciences, Saint Louis Zoo, Saint Louis, MI, USA 2 CREW, Cincinnati Zoo and Botanical Garden, Cincinnati, OH, USA E-mail address: [email protected] (C.S. Asa). Spermatozoa exhibit considerable interspecific variability in size and morphology, but understanding of this diversity is limited. We investigated interspecific variation in nine species and two sub-species representing seven genera in the family Canidae (gray wolf, Canis lupus; Mexican gray wolf, C. l. baileyi; domestic dog C. l. familiaris; red wolf, C. rufus; African painted dog, Lycaon pictus; maned wolf, Chrysocyon brachyurus; bush dog, Speothos venaticus; gray fox, Urocyon cinereoargenteus; red fox, Vulpes vulpes; fennec fox, V. zerda; swift fox, V. velox; bat-eared fox, Otocyon megalotus) nine of which have not been reported previously. Images of fixed sperm were captured using a digital camera (Nikon CoolPix) mounted on a Nikon microscope (Optophot2). A calibration scale using a stage micrometer was also photographed. Head width, length and circumference, midpiece length, and tail length were measured; head area was calculated using Auto-Montage software (Synchroscopy, Frederick, MD, USA). Twenty-five morphologically normal sperm cells were measured per male. There was no consistent relationship between male body size and sperm measures. Sperm of the fennec fox, the smallest canid species (1–2 kg), had the largest head area (23.2 m ± 0.66 SEM), the longest head (6.5 m ± 0.12 SEM) and midpiece (12.1 m ± 0.20 SEM), and secondwidest head (4.1 m ± 0.13 SEM). In contrast, sperm of another small-bodied canid, the bush dog (5–8 kg), had the smallest head area (15.3 m ± 0.82 SEM) and head length (5.2 m ± 0.18 SEM). The medium-sized African wild dog (20–30 kg) had sperm with the widest head (4.2 m ± 0.11 SEM), longest tail (54.9 m ± 0.78 SEM) and longest total sperm length (72.7 m ± 0.78 SEM). However, large-bodied Mexican gray wolves (30–45 kg) had sperm with the shortest midpiece (9.3 m ± 0.40 SEM), tail (40.4 m ± 1.1 SEM) and total sperm length (55.4 m ± 1.4 SEM). Because all canid species have a monogamous mating system, sperm competition would not be expected to drive such divergence in sperm size parameters. However, opportunities for extra-pair mating, related to size of territory or social grouping, vary among species. Thus, sperm competition might have selected for larger sperm components in more social species, such as painted dogs and fennec foxes. http://dx.doi.org/10.1016/j.anireprosci.2016.03.069
G.P. Johnson 1,∗ , A.M. English 1,2 , D.A. Hoey 3 , K.G. Meade 2 , S. Fair 1 1 Department of Life Sciences, University of Limerick, Limerick, Ireland 2 Animal and Bioscience Research Department, Animal and Grassland Research and Innovation Centre, Teagasc, Grange, Dunsany, Meath, Ireland 3 Trinity Centre for Bioengineering, Trinity College, Dublin, Ireland E-mail address: [email protected] (G.P. Johnson).
Cation channel of sperm (CatSper), are sperm-specific calcium channels involved in the regulation of sperm function and male fertility of many species. The aim of this study was to use a comparative genomics approach to identify and characterise the evolutionary orthologs of CatSper genes in the bovine genome. Orthology searches using the basic local alignment search tool (BLAST) was performed for four known human and mouse CatSper gene sequences (CatSper 1-4) in the bovine genome (version: bosTau8). The bioinformatic tool, BLAST-Like Alignment Tool (BLAT), was used to determine the chromosomal position of the four othologous bovine genes identified. Phylogenetic analysis was also performed to investigate the evolutionary relationships between the four novel bovine CatSper genes and their evolutionary orthologs using MEGA software. A multiple sequence alignment for all genes was performed using T-coffee and annotated using Jalview. To characterise the expression profile of these novel genes, bull reproductive tract tissues, including parenchyma testis, rete testis and the different segments of the epididymis (caput, corpus, cauda) were collected from sexually mature beef bulls (n = 4) within 10 min of slaughter. All tissue samples were immediately snap frozen in liquid nitrogen and transported to the laboratory for RNA extraction and cDNA synthesis. Quantitative real-time polymerase chain reaction was performed. Data were analysed using univariate ANOVA (SPSS, v. 22). A significant effect of tissue location was detected for expression of all four of the Catsper genes, with Catsper 1-4 upregulated in the parenchyma testis compared to the three segments of the epididymis (P < 0.01). For CatSper 1-4 the rete testis had significantly higher expression than the caudal and corpus epididymis (P < 0.01) however, no difference in expression level between it and the caput epididymis or the parenchyma testis (P > 0.05) was detected. The caudal epididymis had lower expression of all four CatSper genes than the parenchyma and rete testis (P < 0.01). This study is the first report on the identification and initial characterisation of CatSper genes in the bovine. The testes expression and location-specific changes in mRNA abundance support an evolutionary conserved role for these channels in bull reproduction. http://dx.doi.org/10.1016/j.anireprosci.2016.03.070
Abstracts / Animal Reproduction Science 169 (2016) 99–135
P50
P51
Interspecific divergence of sperm morphometry in canids
Comparative genomic identification and expression profiling of CatSper genes in the reproductive tract of the bull
C.S. Asa 1,∗ , L. Vansandt 2 , K. Bauman 1 1
Reproductive and Behavioral Sciences, Saint Louis Zoo, Saint Louis, MI, USA 2 CREW, Cincinnati Zoo and Botanical Garden, Cincinnati, OH, USA E-mail address: [email protected] (C.S. Asa). Spermatozoa exhibit considerable interspecific variability in size and morphology, but understanding of this diversity is limited. We investigated interspecific variation in nine species and two sub-species representing seven genera in the family Canidae (gray wolf, Canis lupus; Mexican gray wolf, C. l. baileyi; domestic dog C. l. familiaris; red wolf, C. rufus; African painted dog, Lycaon pictus; maned wolf, Chrysocyon brachyurus; bush dog, Speothos venaticus; gray fox, Urocyon cinereoargenteus; red fox, Vulpes vulpes; fennec fox, V. zerda; swift fox, V. velox; bat-eared fox, Otocyon megalotus) nine of which have not been reported previously. Images of fixed sperm were captured using a digital camera (Nikon CoolPix) mounted on a Nikon microscope (Optophot2). A calibration scale using a stage micrometer was also photographed. Head width, length and circumference, midpiece length, and tail length were measured; head area was calculated using Auto-Montage software (Synchroscopy, Frederick, MD, USA). Twenty-five morphologically normal sperm cells were measured per male. There was no consistent relationship between male body size and sperm measures. Sperm of the fennec fox, the smallest canid species (1–2 kg), had the largest head area (23.2 m ± 0.66 SEM), the longest head (6.5 m ± 0.12 SEM) and midpiece (12.1 m ± 0.20 SEM), and secondwidest head (4.1 m ± 0.13 SEM). In contrast, sperm of another small-bodied canid, the bush dog (5–8 kg), had the smallest head area (15.3 m ± 0.82 SEM) and head length (5.2 m ± 0.18 SEM). The medium-sized African wild dog (20–30 kg) had sperm with the widest head (4.2 m ± 0.11 SEM), longest tail (54.9 m ± 0.78 SEM) and longest total sperm length (72.7 m ± 0.78 SEM). However, large-bodied Mexican gray wolves (30–45 kg) had sperm with the shortest midpiece (9.3 m ± 0.40 SEM), tail (40.4 m ± 1.1 SEM) and total sperm length (55.4 m ± 1.4 SEM). Because all canid species have a monogamous mating system, sperm competition would not be expected to drive such divergence in sperm size parameters. However, opportunities for extra-pair mating, related to size of territory or social grouping, vary among species. Thus, sperm competition might have selected for larger sperm components in more social species, such as painted dogs and fennec foxes. http://dx.doi.org/10.1016/j.anireprosci.2016.03.069
G.P. Johnson 1,∗ , A.M. English 1,2 , D.A. Hoey 3 , K.G. Meade 2 , S. Fair 1 1 Department of Life Sciences, University of Limerick, Limerick, Ireland 2 Animal and Bioscience Research Department, Animal and Grassland Research and Innovation Centre, Teagasc, Grange, Dunsany, Meath, Ireland 3 Trinity Centre for Bioengineering, Trinity College, Dublin, Ireland E-mail address: [email protected] (G.P. Johnson).
Cation channel of sperm (CatSper), are sperm-specific calcium channels involved in the regulation of sperm function and male fertility of many species. The aim of this study was to use a comparative genomics approach to identify and characterise the evolutionary orthologs of CatSper genes in the bovine genome. Orthology searches using the basic local alignment search tool (BLAST) was performed for four known human and mouse CatSper gene sequences (CatSper 1-4) in the bovine genome (version: bosTau8). The bioinformatic tool, BLAST-Like Alignment Tool (BLAT), was used to determine the chromosomal position of the four othologous bovine genes identified. Phylogenetic analysis was also performed to investigate the evolutionary relationships between the four novel bovine CatSper genes and their evolutionary orthologs using MEGA software. A multiple sequence alignment for all genes was performed using T-coffee and annotated using Jalview. To characterise the expression profile of these novel genes, bull reproductive tract tissues, including parenchyma testis, rete testis and the different segments of the epididymis (caput, corpus, cauda) were collected from sexually mature beef bulls (n = 4) within 10 min of slaughter. All tissue samples were immediately snap frozen in liquid nitrogen and transported to the laboratory for RNA extraction and cDNA synthesis. Quantitative real-time polymerase chain reaction was performed. Data were analysed using univariate ANOVA (SPSS, v. 22). A significant effect of tissue location was detected for expression of all four of the Catsper genes, with Catsper 1-4 upregulated in the parenchyma testis compared to the three segments of the epididymis (P < 0.01). For CatSper 1-4 the rete testis had significantly higher expression than the caudal and corpus epididymis (P < 0.01) however, no difference in expression level between it and the caput epididymis or the parenchyma testis (P > 0.05) was detected. The caudal epididymis had lower expression of all four CatSper genes than the parenchyma and rete testis (P < 0.01). This study is the first report on the identification and initial characterisation of CatSper genes in the bovine. The testes expression and location-specific changes in mRNA abundance support an evolutionary conserved role for these channels in bull reproduction. http://dx.doi.org/10.1016/j.anireprosci.2016.03.070