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Evaluation of single nucleotide polymorphism of protamine-1 and -2 genes and sperm protamine deficiency in men undergoing ICSI procedure
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reproductive biology 13s (2013) 30–45
of this study was to analyze the in vivo effects of OP on the expression and distribution of adherens and gap junction proteins, N-cadherin, b-catenin and connexin 43 (Cx43), in bank vole testes. Adult males kept in short or long photoperiod were treated with 200 mg/kg body weight of OP for 30 or 60 days. To investigate the localization and expression of cell junction proteins immunohistochemical staining and Western Blot analysis were performed. Plasma FSH concentrations were measured using commercially available Mouse Follitropin subunit b ELISA Kit. We found that in bank vole testes expression and distribution of N-cadherin, b-catenin and Cx43 were dependent on the photoperiod. Long term treatment with OP resulted in the reduction of N-cadherin, b-catenin and Cx43 proteins expression (p<0.05, p<0.01) and their delocalization in the testes of males kept in long photoperiod. On the other hand, in short-day animals slight increase of Cx43 (p<0.05), N-cadherin and bcatenin (statistically non-significant) levels was observed. These differences appeared to be independent on FSH, since OP treatment did not change FSH concentrations. Taken together the results indicate that expression and distribution of adherens and gap junction proteins in seminiferous epithelium of bank vole are photoperiod-dependent. It has also been shown that these proteins are target for OP and their susceptibility to OP is related to photoperiod. http://dx.doi.org/10.1016/j.repbio.2012.11.032 [P22] Assessment of the effect of heterosis on semen parameters of two-breed crosses boars Stanisław Kondracki, Anna Wysokin´ska Department of Animal Reproduction and Hygiene, Institute of Bioengineering and Animal Breeding, Siedlce University of Natural Sciences and Humanities, Siedlce The present work aimed at evaluating the effect of heterosis on semen parameters in two-breed boar hybrids derived from the Duroc, Hampshire and Pietrain breeds. The study was performed on 4609 ejaculates obtained from 32 boars, including 14 purebred and 18 two-breed crosses. The ejaculates were collected manually every 4-5 days. The study involved ejaculates collected from each boar from the first day of its insemination use until the day of semen depletion. Each ejaculate was assessed for the following physical parameters: ejaculate volume, sperm concentration, percentage of spermatozoa with correct motility, total sperm count per ejaculate and number of insemination doses obtained from one ejaculate. The effect of heterosis was calculated on the physical parameters of the boar crosses ejaculates in relation to the mean value of a given parameter for the parent breeds. Positive and well pronounced heterosis effects were identified in the majority of the physical ejaculate parameters of the cross boars for all the cross-breeding variants. A negative effect of heterosis was only identified in the ejaculate sperm concentration of the Hampshire x Pietrain crosses. The highest ejaculatory efficiency was identified in the hybrids produced on the basis of the Hampshire breed. These breeders were also found to feature significant heterosis effects on ejaculate volume, total ejaculate sperm count and number of insemination doses prepared from one ejaculate. http://dx.doi.org/10.1016/j.repbio.2012.11.033 [P23] Evaluation of single nucleotide polymorphism of protamine-1 and -2 genes and sperm protamine deficiency in men undergoing ICSI procedure Anna Kazienko 1,2,*, Anna Rymaszewska 3, Kamil Gill 1, Dariusz Ga˛czarzewicz 4, Maria Laszczyn´ska 1, Rafał Kurzawa 2, Małgorzata Piasecka 1 1 Department of Histology and Biology of Development, Pomerania Medical University, Szczecin 2 Department of Reproductive Medicine and Gynecology, Pomeranian Medical University, Szczecin-Police 3 Department of Genetics, University of Szczecin 4 Department of Animal Reproduction, Biotechnology and Environmental Hygiene, Faculty of Biotechnology and Animal Breeding, West Pomeranian University of Technology, Szczecin *Corresponding author. Genetics factors play an important role in male idiopathic infertility and cause poor outcomes in assisted reproduction. The sperm protamines are responsible for DNA packaging during spermatogenesis. Abnormal condensation of chromatin can result from aberrations of gene protamine expression (PRM1, PRM2) and protamine deficiency. These defects can impair proper fertilization of egg cell and development of the embryo. The aim of our research was to determine the incidence of spermatozoa with abnormal protamination (test with fluorochrome A3 – CMA3) and to find single nucleotide polymorphisms (SNPs) in PRM1 and PRM2 (sequencing of PCR products) in man undergoing ICSI procedure (n = 57) and fertile man (n = 28). No significant difference was found in the proportion of CMA3-positive sperm cells between compared groups (median 20.00% vs. 23.00%). Our study revealed 2 SNP only in PRM1 (c.-190C>A, rs2301365, promoter region; c.139C>A, rs737008, exon 2, synonymous polymorphism, p.Arg47Arg). Rare polymorphic change c.*62G>C in 3’UTR in PRM2 (rs79674436) strongly connected with male factor infertility was not found. In the case of c.-190C>A 33.33% and 25.00% homozygous (CC; p = 0.71), 27.78% and 50.00% heterozygous (CA; p = 0.33) as well as 38.89% and 25.00% homozygous (AA; p = 0.62) type were identified in fertile man and ICSI patients, respectively. In turn of c.139C>A 0.0% and 13.33% CC (p = 0.19), 31.42% and 13.33% CA (p = 0.26), 68.42% and 73.34% AA (p = 1.0) genotype were noted in compared groups. There were no significant differences in the deficiency of protamination and in the frequency of discovered polymorphic changes in PRM1 gene between fertile men and patients underwent ICSI procedure. http://dx.doi.org/10.1016/j.repbio.2012.11.034
reproductive biology 13s (2013) 30–45
of this study was to analyze the in vivo effects of OP on the expression and distribution of adherens and gap junction proteins, N-cadherin, b-catenin and connexin 43 (Cx43), in bank vole testes. Adult males kept in short or long photoperiod were treated with 200 mg/kg body weight of OP for 30 or 60 days. To investigate the localization and expression of cell junction proteins immunohistochemical staining and Western Blot analysis were performed. Plasma FSH concentrations were measured using commercially available Mouse Follitropin subunit b ELISA Kit. We found that in bank vole testes expression and distribution of N-cadherin, b-catenin and Cx43 were dependent on the photoperiod. Long term treatment with OP resulted in the reduction of N-cadherin, b-catenin and Cx43 proteins expression (p<0.05, p<0.01) and their delocalization in the testes of males kept in long photoperiod. On the other hand, in short-day animals slight increase of Cx43 (p<0.05), N-cadherin and bcatenin (statistically non-significant) levels was observed. These differences appeared to be independent on FSH, since OP treatment did not change FSH concentrations. Taken together the results indicate that expression and distribution of adherens and gap junction proteins in seminiferous epithelium of bank vole are photoperiod-dependent. It has also been shown that these proteins are target for OP and their susceptibility to OP is related to photoperiod. http://dx.doi.org/10.1016/j.repbio.2012.11.032 [P22] Assessment of the effect of heterosis on semen parameters of two-breed crosses boars Stanisław Kondracki, Anna Wysokin´ska Department of Animal Reproduction and Hygiene, Institute of Bioengineering and Animal Breeding, Siedlce University of Natural Sciences and Humanities, Siedlce The present work aimed at evaluating the effect of heterosis on semen parameters in two-breed boar hybrids derived from the Duroc, Hampshire and Pietrain breeds. The study was performed on 4609 ejaculates obtained from 32 boars, including 14 purebred and 18 two-breed crosses. The ejaculates were collected manually every 4-5 days. The study involved ejaculates collected from each boar from the first day of its insemination use until the day of semen depletion. Each ejaculate was assessed for the following physical parameters: ejaculate volume, sperm concentration, percentage of spermatozoa with correct motility, total sperm count per ejaculate and number of insemination doses obtained from one ejaculate. The effect of heterosis was calculated on the physical parameters of the boar crosses ejaculates in relation to the mean value of a given parameter for the parent breeds. Positive and well pronounced heterosis effects were identified in the majority of the physical ejaculate parameters of the cross boars for all the cross-breeding variants. A negative effect of heterosis was only identified in the ejaculate sperm concentration of the Hampshire x Pietrain crosses. The highest ejaculatory efficiency was identified in the hybrids produced on the basis of the Hampshire breed. These breeders were also found to feature significant heterosis effects on ejaculate volume, total ejaculate sperm count and number of insemination doses prepared from one ejaculate. http://dx.doi.org/10.1016/j.repbio.2012.11.033 [P23] Evaluation of single nucleotide polymorphism of protamine-1 and -2 genes and sperm protamine deficiency in men undergoing ICSI procedure Anna Kazienko 1,2,*, Anna Rymaszewska 3, Kamil Gill 1, Dariusz Ga˛czarzewicz 4, Maria Laszczyn´ska 1, Rafał Kurzawa 2, Małgorzata Piasecka 1 1 Department of Histology and Biology of Development, Pomerania Medical University, Szczecin 2 Department of Reproductive Medicine and Gynecology, Pomeranian Medical University, Szczecin-Police 3 Department of Genetics, University of Szczecin 4 Department of Animal Reproduction, Biotechnology and Environmental Hygiene, Faculty of Biotechnology and Animal Breeding, West Pomeranian University of Technology, Szczecin *Corresponding author. Genetics factors play an important role in male idiopathic infertility and cause poor outcomes in assisted reproduction. The sperm protamines are responsible for DNA packaging during spermatogenesis. Abnormal condensation of chromatin can result from aberrations of gene protamine expression (PRM1, PRM2) and protamine deficiency. These defects can impair proper fertilization of egg cell and development of the embryo. The aim of our research was to determine the incidence of spermatozoa with abnormal protamination (test with fluorochrome A3 – CMA3) and to find single nucleotide polymorphisms (SNPs) in PRM1 and PRM2 (sequencing of PCR products) in man undergoing ICSI procedure (n = 57) and fertile man (n = 28). No significant difference was found in the proportion of CMA3-positive sperm cells between compared groups (median 20.00% vs. 23.00%). Our study revealed 2 SNP only in PRM1 (c.-190C>A, rs2301365, promoter region; c.139C>A, rs737008, exon 2, synonymous polymorphism, p.Arg47Arg). Rare polymorphic change c.*62G>C in 3’UTR in PRM2 (rs79674436) strongly connected with male factor infertility was not found. In the case of c.-190C>A 33.33% and 25.00% homozygous (CC; p = 0.71), 27.78% and 50.00% heterozygous (CA; p = 0.33) as well as 38.89% and 25.00% homozygous (AA; p = 0.62) type were identified in fertile man and ICSI patients, respectively. In turn of c.139C>A 0.0% and 13.33% CC (p = 0.19), 31.42% and 13.33% CA (p = 0.26), 68.42% and 73.34% AA (p = 1.0) genotype were noted in compared groups. There were no significant differences in the deficiency of protamination and in the frequency of discovered polymorphic changes in PRM1 gene between fertile men and patients underwent ICSI procedure. http://dx.doi.org/10.1016/j.repbio.2012.11.034