CRISPR/Cas9-mediated genome editing reveals 12 testis-enriched genes dispensable for male fertility in mice / 亚洲男科学杂志(英文版)

Gene expression analyses suggest that more than 1000-2000 genes are expressed predominantly in mouse and human testes. Although functional analyses of hundreds of these genes have been performed, there are still many testis-enriched genes whose functions remain unexplored. Analyzing gene function using knockout (KO) mice is a powerful tool to discern if the gene of interest is essential for sperm formation, function, and male fertility in vivo. In this study, we generated KO mice for 12 testis-enriched genes, 1700057G04Rik, 4921539E11Rik, 4930558C23Rik, Cby2, Ldhal6b, Rasef, Slc25a2, Slc25a41, Smim8, Smim9, Tmem210, and Tomm20l, using the clustered regularly interspaced short palindromic repeats /CRISPR-associated protein 9 (CRISPR/Cas9) system. We designed two gRNAs for each gene to excise almost all the protein-coding regions to ensure that the deletions in these genes result in a null mutation. Mating tests of KO mice reveal that these 12 genes are not essential for male fertility, at least when individually ablated, and not together with other potentially compensatory paralogous genes. Our results could prevent other laboratories from expending duplicative effort generating KO mice, for which no apparent phenotype exists.

Prevalence of the MTHFR C677T Mutation in Fertile and Infertile Women / Prevalência da mutação MTHFR C677T em mulheres férteis e inférteis

Abstract Introduction The importance of the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene in infertile women remains controversial. Objective To evaluate if the MTHFR C677T mutations are more frequent in infertile women, and if they can be associated with the occurrence of infertility in the Brazilian population. Methods This case-control study included 130 infertile women consulting at a private clinic betweenMarch 2003 andMarch 2005 (data previously published), and 260 fertile women attending the family planning outpatient clinic of our institution between April 2012 and March 2013. Data analysis The Chi-squared and Fisher Exact tests were used to evaluate the association between the presence of the MTHFR C677T mutation and a history of infertility. Results The frequency of the mutation was of 58.5% for the case group (n = 76) and of 49.2% for the fertile controls (n = 128). The mutation was homozygous in 13 women in the case group (10%) and in 23 of the fertile women in the control group (8.8%). These differences were not statistically significant. Conclusions These results suggest that the presence of the MTHFR C677T mutation does not constitute a risk factor for infertility, even when themutation is homozygous. Further studies are needed to confirm whether research on this mutation should be considered unnecessary in women with infertility.

Resumo Introdução A importância da mutação C677T no gene da metilenotetrahidrofolato redutase (MTHFR) em mulheres com infertilidade permanece controversa. Objetivo Avaliar se a mutação MTHFR C677Témais frequente em mulheres inférteis, e se pode ser associada com a ocorrência de infertilidade na população brasileira. Métodos Estudo de caso-controle, com avaliação de 130 mulheres com infertilidade atendidas em clínica privada no período de março de 2003 a março de 2005 (dados previamente publicados) e 260 mulheres férteis atendidas no ambulatório de planejamento familiar de nossa instituição no período de abril de 2012 a março de 2013. Análise dos dados Foram utilizados os testes de Qui-quadrado e Exato de Fisher para o estudo da associação entre a presença damutação MTHFR C677T e o antecedente de infertilidade. Resultados A frequência da mutação foi de 58,5% nos casos (n = 76) e de 49,2% nos controles (n = 128). Dentre os casos, 13 apresentavam esta mutação em homozigose (10%). Nos controles, a homozigose foi encontrada em 23 mulheres férteis (8,8%). Estas diferenças não foram estatisticamente significativas. Conclusões Este estudo sugere que a presença da mutação MTHFR C677T não constitui fator de risco para infertilidade, mesmo em casos de homozigose. Estudos complementares são necessários para ratificar se a investigação desta mutação deve ser considerada desnecessária em mulheres com infertilidade.

Análise genética de características de fertilidade, de crescimento e de produtividade em vacas da raça Canchim / Genetic analysis of fertility, growth and productivity traits of Canchim cows

O objetivo deste trabalho foi estimar herdabilidades e correlações genéticas para idade (IPP), peso (PPP) e escore da condição corporal (EPP) ao primeiro parto, tempo de permanência no rebanho (TPR), número (ND10) e quilograma (QD10) de bezerros desmamados em até dez anos de idade, número (NDT) e quilograma (QDT) de bezerros desmamados durante a permanência no rebanho, quilograma de bezerros desmamados por ano de permanência no rebanho (QTPR), peso adulto (PAD) e parâmetros A e k da curva de crescimento de Bertalanffy de fêmeas de um rebanho da raça Canchim. As distribuições a posteriori dos componentes de (co)variância foram obtidas por inferência bayesiana, em análises bicaráter de IPP, PPP e EPP com as outras características. Os modelos estatísticos incluíram os efeitos aleatórios genético aditivo direto e residual e os efeitos fixos de ano e mês de nascimento ou do parto e idade da vaca ao parto, dependendo da característica. As médias a posteriori das herdabilidades foram 0,12 (IPP); 0,51 (PPP); 0,36 (EPP); 0,22 (TPR); 0,24 (ND10); 0,24 (QD10); 0,25 (NDT); 0,23 (QDT); 0,31 (QTPR); 0,56 (PAD); 0,60 (A) e 0,54 (k), indicando que as características possuem variação genética aditiva suficiente para apresentar boa resposta à seleção massal, com exceção de IPP. As correlações genéticas de TPR (-0,35; -0,23 e -0,02), ND10 (-0,33; -0,40 e -0,02), QD10 (-0,47; -0,29 e 0,00), NDT (-0,43; -0,41 e -0,02), QDT (-0,46; -0,28 e 0,00), QTPR (-0,52; -0,35 e 0,00), PAD (0,19; 0,86 e 0,40), A (0,07; 0,09 e -0,18) e k (-0,21; 0,08 e 0,04) com IPP, PPP e EPP, respectivamente, sugerem que a seleção para reduzir a IPP deve melhorar a longevidade e as características de produtividade das fêmeas; porém, o aumento do PPP poderá prejudicá-las...

The objective of this work was to estimate heritabilities and genetic correlations for age at first calving (AFC), weight at first calving (WFC), body condition score at first calving (SFC), culling age (DIH, days in herd), number (NW10) and kilograms (KW10) of calves weaned up to ten years of age, total number (TNW) and total kilograms (TQW) of calves weaned during herd life, kilograms of calves weaned per year in herd (KYH), adult body weight (ABW) and parameters A and k of the Bertalanffy growth curve of females of a Canchim (5/8 Charolais + 3/8 Zebu) beef cattle herd. The variance and covariance components were obtained by two-trait analyses of AFC, WFC and SFC with the other production traits, using the bayesian inference with statistical models that included the additive direct and residual random effects, and the fixed effects of year and month of birth or of calving and calving age, depending on the trait. The posteriori means of the heritability estimates were 0.12 (AFC), 0.51 (WFC), 0.36 (SFC), 0.22 (DIH), 0.24 (NW10), 0.24 (KW10), 0.25 (TNW), 0.23 (TKW), 0.31 (KYH), 0.56 (ABW), 0.60 (A) and 0.54 (k), indicating that these traits have enough additive genetic variation to show response to mass selection. The genetic correlations for DIH (-0.35, -0.23 and -0.02), NW10 (-0.33, -0.40 and -0.02), KW10 (-0.47, -0.29 and 0.00), TNW (-0.43, -0.41 and -0.02), TKW (-0.46, -0.28 and 0.00), KYH (-0.52, -0.35 and 0.00), ABW (0.19, 0.86 and 0.40), A (0.07, 0.09 and -0.18) and k (-0.21, 0.08 and 0.04) with AFC, WFC and SFC, suggest that selection to reduce age at first calving should improve longevity and productivity traits of females, but the increase in body weight at first calving should reduce these traits...

Mating latency, duration of copulation and fertility in four species of the Drosophila bipectinata complex.

Significant interspecific variations in mean duration of copulation and fertility were observed in four species of the Drosophila bipectinata species complex. However, D. bipectinata showed positive correlation between duration of copulation and fertility. Similarly, D. malerkotliana showed negative correlation between mating latency and duration of copulation. Likewise, D. pseudoananassae showed positive correlation between mating latency and fertility. These results suggest that D. pseudoananassae has distant relatedness from the other three species with respect to mating latency, duration of copulation and fertility which supports the previous findings.

Electrolyzed-reduced water increases resistance to oxidative stress, fertility, and lifespan via insulin/IGF-1-like signal in C. elegans

Electrolyzed-reduced water (ERW) scavenges reactive oxygen species and is a powerful anti-oxidant. A positive correlation between oxidative stress and aging has been proved in many model organisms. In Caenorhabditis elegans, many long-lived mutants showed reduced fertility as a trade off against longevity phenotype. We aimed to study the effect of ERW on oxidative stress, fertility and lifespan of C. elegans. We also investigated the genetic pathway involved in the effect of ERW on resistance to oxidative stress and lifespan. We compared lifespan and fertility of worms in media prepared with distilled water and ERW. ERW significantly extended lifespan and increased the number of progeny produced. Then the effect of ERW on resistance to oxidative stress and lifespan of long-lived mutants was determined. ERW increased resistance to oxidative stress and lifespan of eat-2, a genetic model of dietary restriction, but had no effect on those of age-1, which is involved in insulin/insulin-like growth factor (IGF)-1-like signal. In addition, knockdown of daf-16, the downstream mediator of insulin/IGF-1-like signal, completely prevented the effect of ERW on lifespan. These findings suggest that ERW can extend lifespan without accompanying reduced fertility and modulate resistance to oxidative stress and lifespan via insulin/IGF-1-like signal in C. elegans.

Age at menarche and related factors in girlsof urban areas of Markazi (Central) province of Iran / Edad de la menarquia y factores relacionados en las niñas de zonas urbanas de la provincia Markazi (Central) de Irán

The age at menarche is an indicator of racial, geographical and nutritional patterns of different societies. This cross-sectional study conducted on 1223 girls aged 14-20 in the Markazi (Central) Province, Iran in 2010. In this research, the age at menarche, as the main variable, was determined for each city and compared between different cities. The statistical analyses included c2 test for determining statistically significant differences, variance analysis for determining the difference between groups, and Pearson correlation coefficient for determining the relationship between variables. The average age of first menstruation was 13.21+/-1.33 years in the Markazi Province, with no significant difference among the cities of the province. This age is inversely related to body mass index (BMI), nutritional status and weight. The pattern of distribution of BMI is similar for all the cities in the province. Although, the average age at menarche in the Markazi (central) Province, a cold area, is higher compared to warmer regions in Iran, it may be more influenced by race than by the climate. Considering the lower age at menarche in Markazi Province compared to previous similar studies, it may be stated that social well-being and nutritional standards have improved in the province.

La edad de la menarquia es un indicador de patrones raciales, geográficos y nutricionales de distintas sociedades. Este estudio transversal fue realizado el 2010 en 1223 niñas entre 14 y 20 años en la Provincia Markazi (Central), Irán. En esta investigación, la edad de la menarquia, como la variable principal, se determinaron para cada ciudad y en comparación entre las diferentes ciudades. Los análisis estadísticos incluyeron la prueba c2 para determinar diferencias estadísticamente significativas, el análisis de varianza para determinar la diferencia entre los grupos, y el coeficiente de correlación de Pearson para determinar la relación entre las variables. El promedio de edad de la primera menstruación fue 13,21+/-1,33 años en la provincia de Markazi, sin diferencias significativas entre las ciudades de la provincia. Esta edad es inversamente proporcional al índice de masa corporal (IMC), el estado nutricional y el peso. El patrón de distribución del IMC es similar para todas las ciudades de la provincia. Aunque la edad media de la menarquia en la Provincia Markazi (central), una zona de clima frío, es mayor en comparación con las regiones más cálidas en Irán, puede estar más influenciadas por la raza que por el clima. Teniendo en cuenta la baja edad de menarquia en la Provincia Markazi en comparación con estudios similares anteriores, se puede afirmar que el bienestar social y los niveles de nutrición han mejorado en la provincia.

Opportunity for natural selection among some selected population groups of Northeast India.

BACKGROUND: Selection potential based on differential fertility and mortality has been computed for seven population groups inhabiting different geographical locations of Northeast India. MATERIALS AND METHODS: Crow’s as well as Johnston and Kensinger’s index have been used for the present purpose. RESULTS AND CONCLUSION: Irrespective of the methodology, the total index of selection was found to be highest among the Deoris followed by the Kaibartas. The lowest selection index was found among the Oraon population. If the relative contribution of fertility and mortality components to the total index is considered to be multiplicative, it is observed that in all these communities the fertility component exceeds that of mortality component, which may indicate initiation of demographic transitional phase in the selected populations with the betterment of healthcare and socioeconomic condition within the last few decades.

Reproductive relationships and degree of synapsis in the polytene chromosomes of the Drosophila buzzatii species cluster

O processo de especiação ocorre pela evolução de qualquer uma das diversas formas de isolamento reprodutivo entre táxons, incluindo inviabilidade de híbridos. Neste trabalho, linhagens provenientes de populações alopátricas de espécies de Drosophila do cluster buzzatii foram cruzadas experimentalmente com o objetivo de avaliar suas relações reprodutivas e citogenéticas, e contribuir para o entendimento do isolamento reprodutivo neste grupo de espécies intimamente relacionadas. Os cruzamentos dentro de uma mesma linhagem foram altamente férteis, contudo as diferenças na fertilidade e fecundidade encontradas intra e interespecificamente são discutidas neste artigo. Dos 30 cruzamentos interespecíficos, 63% foram parcialmente ou completamente estéreis. Cinqüenta e três por cento dos cruzamentos de F1 interespecíficos (fêmeas e machos F1 cruzados entre si) também foram parcialmente ou completamente estéreis, em contraste com apenas um, em 24 cruzamentos de F1 intra-específicos, que foi parcialmente estéril. A análise dos cromossomos politênicos nos híbridos revelou sinapse completa, exceto nos microcromossomos (VI) e na região proximal do cromossomo X. A divergência intra-específica e o grau variável de pareamento cromossômico, observados neste estudo, revelam parte da complexidade do processo de especiação pertinente ao cluster buzzatii, o que é consistente com diferentes caracteres estudados neste cluster.

Frequency of the allelic variant (Trp8Arg/Ile15Thr) of the luteinizing hormone gene in a Brazilian cohort of healthy subjects and in patients with hypogonadotropic hypogonadism

OBJETIVO: Avaliar a freqüência da variante alélica (Trp8Arg/Ile15Thr) do gene da subunidade b do hormônio luteinizante em um grupo de brasileiros saudáveis e em pacientes portadores de hipogonadismo hipogonadotrófico.CASUÍSTICA E MÉTODOS: Duzentos e dois adultos (115 mulheres) com função sexual preservada e 48 pacientes (24 mulheres) portadoras de hipogonadismo hipogonadotrófico foram submetidos a estudo molecular utilizando técnicas de reação em cadeia da polimerase seguida por digestão enzimática com as enzimas de restrição Nco I (para detecção da mutação pontual Trp8Arg) e Fok I (para detecção da mutação pontual Ile15Thr). Os níveis basais de hormônio luteinizante e FSH, testosterona ou estradiol foram dosados em 37 indivíduos normais (21 mulheres) e 27 pacientes portadores de hipogonadismo hipogonadotrófico (13 mulheres) pelo método imunofluorométrico (hLH-Spec and hFSH-Spec, AutoDELFIA, Wallac Oy, Turku, Finland).RESULTADOS: A variante alélica (Arg8/Thr15) do gene da subunidade b do LH apresentou freqüência similar nos indivíduos saudáveis (14.4%) e nos pacientes portadores de hipogonadismo hipogonadotrófico (16.6%). Não houve interferência da variante alélica do gene da subunidade b do LH nos níveis de LH dos indivíduos normais e dos pacientes portadores de hipogonadismo hipogonadotrófico.CONCLUSÃO: Este estudo indica que a variante alélicaArg8/Thr15 do gene da subunidade b do LH é um polimorfismo comum na população brasileira (14.4%). A freqüência similar dessa variante em indivíduos saudáveis e em portadores de hipogonadismo hipogonadotrófico exclui o papel da variante na etiologia do hipogonadismo hipogonadotrófico.

The Hsp60C gene in the 25F cytogenetic region in Drosophila melanogaster is essential for tracheal development and fertility.

Earlier studies have shown that of the four genes (Hsp60A, Hsp60B, Hsp60C, Hsp60D genes) predicted to encode the conserved Hsp60 family chaperones in Drosophila melanogaster, the Hsp60A gene (at the 10A polytene region) is expressed in all cell types of the organism and is essential from early embryonic stages, while the Hsp60B gene (at 21D region) is expressed only in testis, being essential for sperm individualization. In the present study, we characterized the Hsp60C gene (at 25F region), which shows high sequence homology with the other three Hsp60 genes of D. melanogaster. In situ hybridization of Hsp60C-specific riboprobe shows that expression of this gene begins in late embryonic stages (stage 14 onwards), particularly in the developing tracheal system and salivary glands; during larval and adult stages, it is widely expressed in many cell types but much more strongly in tracheae and in developing and differentiating germ cells. A P-insertion mutant (Hsp60C(1)) allele with the P transposon inserted at -251 position of the Hsp60C gene promoter was generated. This early larval recessive lethal mutation significantly reduces levels of Hsp60C transcripts in developing tracheae and this is associated with a variety of defects in the tracheal system, including lack of liquid clearance. About 10% of the homozygotes survive as weak, shortlived and completely sterile adults. Testes of the surviving mutant males are significantly smaller, with fewer spermatocytes, most of which do not develop beyond the round spermatid stage. In situ and Northern hybridizations show significantly reduced levels of the Hsp60C transcripts in Hsp60C(1) homozygous adult males. The absence of early meiotic stages in the Hsp60C(1) homozygous testes contrasts with the effect of testis-specific Hsp60B (21D) gene, whose mutation affects individualization of sperm bundles later in spermiogenesis. In view of the specific effects in tracheal development and in early stages of spermatogenesis, it is likely that, besides its functions as a chaperone, Hsp60C may have signalling functions and may also be involved in cation transport across the developing tracheal epithelial cells.

Análise genética de características ponderais e reprodutivas de fêmeas bovinas Tabapuã / Genetics analysis of growth and reproduction traits in a herd of Tabapuã females

Foram estudadas 1251 informações referentes aos pesos às idades-padrão de 205 (P205), 365 (P365) e 550 (P550) dias e idade ao primeiro parto (IPP) e primeiro intervalo de partos (IEP1) de fêmeas bovinas da raça Tabapuã. As médias, desvios-padrão e coeficientes de variação foram respectivamente: P205 173,9kg, 16,0kg e 9,2; P365 - 225,7kg, 28,7kg e 12,7; P550 - 277,0kg, 36,7kg e 13,3; IPP - 1125,5 dias, 106,6 dias e 9,5 e IEPI - 522,2 dias, 106,2 dias e 20,7. As estimativas de componentes de ( co )variância e de parâmetros genéticos foram obtidas pelo método de máxima verossimilhança restrita, utilizando-se o software MTDFREML. Para cada característica as estimativas de herdabilidade foram de 0,16, 0,25, 0,29, 0,03 e 0,01, na mesma ordem de citação acima. As correlações genéticas entre as características ponderais e reprodutivas foram: -0,67 (p205xIPP); -0,07 (P365xIPP); -0,41 (p550xIPP); 0,19 (p205xIEPl); 0,79 (p365xIEPI) e 0,64 (p550xIEPI).

Endogamy, consanguinity and community genetics.

The population of India is composed of many thousands of subpopulations, divided by geography, language, religion and caste or biraderi (patrilineage) boundaries, with endogamous marriage the norm. The net effect has been the creation of multiple genetic isolates with individual mutation profiles, but to date the clinical consequences of this highly complex differentiation have been largely ignored. In contrast, the topic of consanguinity continues to attract attention among medical and population geneticists, clinicians and social scientists. The significant progress made in India in improving childhood nutritional status and combating infectious disease means that genetic disorders have assumed ever-increasing importance. In populations where consanguineous marriage is widely practised, recessive genetic disorders will continue to gain greater prominence in the overall spectrum of ill health. At the same time this increase will in part be negated by urbanization and the move to smaller family sizes, which predictably will result in a decline in the prevalence of consanguineous unions. Developing an understanding of these changes will require a wide-ranging and multidisciplinary investigative approach for which community genetics is ideally suited.

Male sterility associated with overexpression of the noncoding hsromega gene in cyst cells of testis of Drosophila melanogaster.

Of the several noncoding transcripts produced by the hsromega gene of Drosophila melanogaster, the nucleus-limited >10-kb hsromega-n transcript colocalizes with heterogeneous nuclear RNA binding proteins (hnRNPs) to form fine nucleoplasmic omega speckles. Our earlier studies suggested that the noncoding hsromega-n transcripts dynamically regulate the distribution of hnRNPs in active (chromatin bound) and inactive (in omega speckles) compartments. Here we show that a P transposon insertion in this gene's promoter (at -130 bp) in the hsromega05421; enhancer-trap line had no effect on viability or phenotype of males or females, but the insertion-homozygous males were sterile. Testes of hsromega05421; homozygous flies contained nonmotile sperms while their seminal vesicles were empty. RNA:RNA in situ hybridization showed that the somatic cyst cells in testes of the mutant male flies contained significantly higher amounts of hsromega-n transcripts, and unlike the characteristic fine omega speckles in other cell types they displayed large clusters of omega speckles as typically seen after heat shock. Two of the hnRNPs, viz. HRB87F and Hrb57A, which are expressed in cyst cells, also formed large clusters in these cells in parallel with the hsromega-n transcripts. A complete excision of the P transposon insertion restored male fertility as well as the fine-speckled pattern of omega speckles in the cyst cells. The in situ distribution patterns of these two hnRNPs and several other RNA-binding proteins (Hrp40, Hrb57A, S5, Sxl, SRp55 and Rb97D) were not affected by hsromega mutation in any of the meiotic stages in adult testes. The present studies, however, revealed an unexpected presence (in wild-type as well as mutant) of the functional form of Sxl in primary spermatocytes and an unusual distribution of HRB87F along the retracting spindle during anaphase telophase of the first meiotic division. It appears that the P transposon insertion in the promoter region causes a misregulated overexpression of hsromega in cyst cells, which in turn results in excessive sequestration of hnRNPs and formation of large clusters of omega speckles in these cell nuclei. The consequent limiting availability of hnRNPs is likely to trans-dominantly affect processing of other pre-mRNAs in cyst cells. We suggest that a compromise in the activity of cyst cells due to the aberrant hnRNP distribution is responsible for the failure of individualization of sperms in hsromega05421; mutant testes. These results further support a significant role of the noncoding hsromega-n transcripts in basic cellular activities, namely regulation of the availability of hnRNPs in active (chromatin bound) and inactive (in omega speckles) compartments.

Estimación de Algunos Efectos Genéticos de la Trucha Arcoiris (Oncorhynchus Mikiss) a Partir de un Cruzamiento Dialélico Completo de dos Líneas: I. Fertilidad y Supervivencia / Valuation of some genetic effects of rainbow trout (Oncorhynchus mikiss) from a complete diallelic cross of two lines: I. Fertility and viability

El presente estudio se realizó en el Centro Acuícola "El Zarco", Estado de México, México, para evaluar el comportamiento reproductivo de dos líneas de truchas Arcoiris (Oncorhynchus mikiss), la línea Zarco ("Z") y la línea Jilotzingo ("J"). Con el propósito de estimar efectos genéticos, se utilizó el cruzamiento dialélico, que permite la mediación de la respuesta a la heterosis y a la habilidad combinatoria general y específica, entre las diferentes líneas utilizadas y cruzadas recíprocas. Al evaluar la incubación, fertilidad, viabilidad y moralidad en las etapas de alevinaje y crianza, se encontró una mejor fertilidad para la línea Jilotzingo; la mortalidad presentada en la etapa de alevinaje fue más elevada en los grupos genéticos híbridos (12.65 por ciento) respecto de los puros (2.6 por ciento). La mortalidad durante la etapa de crianza fue mayor en el grupo genético Z x J (6.5 por ciento) con respecto a los otros. Los resultados muestran que no existe heterosis para estas características y que es importante seleccionar líneas maternas con el fin de incrementar la fertilidad

Nuevos conseptos en espermiologia / New concepts in spermatology

Se establecen paràmetros, tècnicas y fòrmulas para una valoraciòn exacta del estudio animal y concomitantemente la forma de valorar de fertilidad masculina. Dicha mètadologia es la usada por el autor laboratorio del I.G.H.