Separation and forensic identification of sperm from cell mixtures using anti-hLCN6 monoclonal antibody coupled magnetic beads / 生物工程学报

Human lipocalin 6 (hLCN6) is an epididymis-specific secretory protein. It binds to sperm and plays important role in sperm maturation. To explore the feasibility for isolating spermatozoa from mixed cells using anti-hLCN6 monoclonal antibody-conjugated immunomagnetic beads (anti-hLCN6 IMBs) and establish a new method for the separation of sperms from mixed stains, 2 sets of 30 cases of cell mixture suspensions and stains containing different proportions of sperm and epithelial cells were prepared. Biotin-labeled anti-hLCN6 monoclonal antibody (mAb) was incubated with the cell mixtures, and the spermatozoa were then isolated with avidin-coated IMBs. Sperm DNA was extracted and analyzed by PCR-STR typing. Differential lysis was also conducted to compare the effect of the two different isolation methods. The dissociation constant (Kd) of anti-hLCN6 mAb was 3.47×10⁻⁹ mol/L measured by ELISA. Western blotting and immunofluorescence assays showed that hLCN6 was detectable on sperm cells and mainly located on the post-acrosomal region of the sperm head, but not in epithelial cells. Anti-hLCN6 IMBs could capture and separate the sperm cells successfully. Microscopic observation showed that the IMBs could bind to the head of sperm specifically. The success rate of STR typing (more than 13 STR loci, RFU>200) was 90% when the number of sperm cells was 10³/mL and 100% when the sperm cells number was equal to or more than 10⁴/mL. When the number of sperm cells was 10³/mL, 10⁴/mL and 10⁵/mL in mixed stain samples, the success rate of STR typing were 40%, 90% and 100%, respectively. Taken together, the anti-hLCN6 immunomagnetic beads (IMB) method described here could be effective for the isolation of sperm from mixed cells, and the success rate was higher than that of the traditional differential lysis strategy. IMB sorting is a simple and efficient method for the separation of sperms from sperm and epithelial cell mixture, and can be utilized as a supplementary method for forensic mixture samples analysis in sexual assault cases.

Nitrosative stress in human spermatozoa causes cell death characterized by induction of mitochondrial permeability transition-driven necrosis / 亚洲男科学杂志(英文版)

Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosis in somatic cells. Peroxynitrite-induced nitrosative stress has emerged as a major cause of impaired sperm function; however, its ability to trigger cell death has not been described in human spermatozoa. The objective here was to characterize biochemical and morphological features of cell death induced by peroxynitrite-mediated nitrosative stress in human spermatozoa. For this, spermatozoa were incubated with and without (untreated control) 3-morpholinosydnonimine (SIN-1), in order to generate peroxynitrite. Sperm viability, mitochondrial permeability transition (MPT), externalization of phosphatidylserine, DNA oxidation and fragmentation, caspase activation, tyrosine nitration, and sperm ultrastructure were analyzed. The results showed that at 24 h of incubation with SIN-1, the sperm viability was significantly reduced compared to untreated control (P < 0.001). Furthermore, the MPT was induced (P < 0.01) and increment in DNA oxidation (P < 0.01), DNA fragmentation (P < 0.01), tyrosine nitration (P < 0.0001) and ultrastructural damage were observed when compared to untreated control. Caspase activation was not evidenced, and although phosphatidylserine externalization increased compared to untreated control (P < 0.001), this process was observed in <10% of the cells and the gradual loss of viability was not characterized by an important increase in this parameter. In conclusion, peroxynitrite-mediated nitrosative stress induces the regulated variant of cell death known as MPT-driven necrosis in human spermatozoa. This study provides a new insight into the pathophysiology of nitrosative stress in human spermatozoa and opens up a new focus for developing specific therapeutic strategies to better preserve sperm viability or to avoid cell death.

Viabilidade dos espermatozoides criopreservados de touros, colhidos do epidídimo após 12 horas a 4ºC / Viability of cryopreserved bull spermatozoa collected from the epididymis stored for 12 hours at 4ºC

O objetivo deste estudo foi avaliar o efeito da refrigeração do epidídimo, sobre a viabilidade dos espermatozoides congelados. Foram colhidos dez pares de testículos/epidídimos de um abatedouro comercial. Um dos epidídimos foi refrigerado a 4°C durante 12 horas e o contralateral foi imediatamente processado. O epidídimo foi isolado, as células espermáticas extraídas e analisadas quanto à motilidade, vigor, concentração, morfologia e integridade da membrana. Após a análise, os espermatozoides foram congelados em diluente Botubov® (Botupharma Biotecnologia Animal, Botucatu, SP, Brasil) e descongelados para análise. O mesmo procedimento foi realizado com o testículo/epidídimo refrigerado. Os resultados evidenciaram maior viabilidade (p≤0,05) das células pré-congelação para os parâmetros, número total de espermatozides (1,9 ± 1,2 versus 0,9 ± 0,9 x 109 espermatozoides), motilidade (74,0 ± 15,1 versus 20,5 ± 13,8%) e vigor (3,7 ± 0,5 versus 1,7 ± 0,8), e pós-congelação, motilidade (23,5 ± 16,7 versus 8,0 ± 7,9%) e vigor (2,0 ± 0,8 versus 0,8 ± 0,8) quando os espermatozoides foram colhidos a partir de epidídimos processados imediatamente após o abate quando comparados aos mantidos 12 horas sob refrigeração, respectivamente. Conclui-se que 12 horas de refrigeração do epidídimo após o abate, prejudica a qualidade das células espermáticas, impossibilitando a congelação do sêmen

The purpose of this study was to evaluate effect of epididymis cooling on bovine frozen sperm viability. Ten pairs of testes/epididymes were collected of a commercial slaughterhouse; one epididymis from each pair was refrigerated at 4ºC for 12 hours and the other immediately proceeded. Epididymis was isolated, sperm cells collected after epididymal slicing and then analyzed regarding to motility, vigor, total number of sperm, morphology and membrane integrity. Sperm cells were frozen in Botubov® extender (Botupharma Biotecnologia Animal, Botucatu, SP, Brazil) and thawed for semen analysis. The same procedure was performed with cooled testis/epididymis. Results demonstrated higher viability (p≤0,05) of fresh cells to the total number of spermatozoa (1,9 ± 1,2 versus 0,9 ± 0,9 x 109 spermatozoa), sperm motility (74,0 ± 15,1 versus 20,5 ± 13,8%) and vigor (3,7 ± 0,5 versus 1,7 ± 0,8), and for pos-thawing motility (23,5 ± 16,7 versus 8,0 ± 7,9%) and vigor (2,0 ± 0,8 versus 0,8 ± 0,8) when spermatozoa were collected immediately post-slaughter than maintained cooling 12 hours, respectively. We conclude that 12 hours of epididymis cooling after slaughter decreases sperm cells quality.

The Toxic Effects of Lead on Testicular Macrophage Immunomodualation and Sperm Cell Parameters in Mice.

Aims: In the present study the toxic effects of lead was investigated experimentally on the testicular macrophages and sperm cells isolated from testes of adult male mice to ascertain the extent of immunomodulation and reproductive dysfunctions (in-vivo). Study Design: Experimental study. Place and Duration of Study: Department of Biotechnology, Assam University, Silchar, Assam, India, between March 2013 and August 2014. Methodology: Dose response study was carried out with an increasing concentration of lead acetate. Percent mortality was determined for these doses and plotted graphically against the respective doses. From the graphs, LD50 value was determined. To validate immunomodulation of testicular macrophages and reproductive dysfunction due to lead intoxication, mice were divided into two groups. One group is treated with lead acetate (10 mg/kg body weight) and the other group with isotonic saline solution for 15 days. The isolated testicular macrophages were used to study the phagocytic property, alteration of enzyme release, cytokine release assay and the sperm cell were used for studying the sperm parameters in both control and treated group. Results: From the study significant decrease in phagocytic index (25516.61±1352.69 to 5154.67±437.37), myeloperoxidase release (77.3±10.7μM to 23.6±4.9μM), nitric oxide release (9.2±1.13 to 4.7±1.69) and a concomitant rise in the pro-inflammatory cytokine TNF-α were observed. These leads to an increased oxidative stress and inflammatory damage in the testes and subsequently less sperm count (78±1.155 to 24.33±1.764), sperm motility and abnormal sperm morphology was documented. Conclusion: Thus it could be concluded that the toxic potential of lead diminished the functional capacities of testicular macrophages, led to immunomodulation and inflammatory damage in testes and thus impede the sperm function parameters, which bear particular significance in heavy metal induced immune infertility in male.

Sperm concentration on the intrauterine artificial insemination in swine / Concentrações espermáticas na inseminação artificial intra-uterina suína

The aim of this work was to evaluate the efficiency of the intrauterine insemination (IUI) in swine, considering the conception rate, farrowing rate, litter size (alive born pigs). For the IUI, the females had been insemination at 24 and 48 hours after the estrus detection, and the inseminating doses of 500 million, 1 billion, 1.5 billion and 2 billion spermatozoa in 20 mL extender had been used. The procedure of catheter insertion through the cervical canal was successfully performed in 97.9 percent of the females. The conception rate was 6.3 percent in the IUI. The farrowing rate in IUI was 87.2 percent but the farrowing rate was 100 percent for the sperm concentration of 500 million. Regarding the number of born pigs and alive born pigs observed in females inseminated with IUI, no significant difference was observed (p > 0.05). The concentration of 500 x 10(6) spermatozoa in 20 mL extender in the intrauterine insemination resulted in an optimal reproductive performance.

Conduziu-se este estudo, com o objetivo de avaliar a eficiência da inseminação intra-uterina (IIU) em suínos, considerando as taxas de retorno ao estro, aborto, parto, além do tamanho da leitegada (número de leitões nascidos e nascidos vivos). Na IIU, as fêmeas foram inseminadas nos tempos de 24 e 48 horas após a detecção do estro, utilizando-se as concentrações de 500 milhões, 1 bilhão, 1,5 bilhão e 2 bilhões de espermatozóides, em 20mL de diluente. A passagem do cateter de IIU através da cérvix foi possível em 97,9 por cento das fêmeas. Foi realizado diagnóstico de retorno ao estro a partir do 18º dia e diagnóstico de gestação por ultrassonografia transcutânea entre o 28º e 30º dias após a inseminação. A taxa de retorno ao estro foi de 6,3 por cento na IIU. A taxa de parto na IIU foi de 87,2 por cento, sendo a taxa de parto para a concentração de 500 milhões de 100 por cento. Com relação ao número de leitões nascidos totais e nascidos vivos, não houve diferenças, entre as diferentes concentrações espermáticas (P>0,05). A utilização da concentração de 500 x 10(6) espermatozóides em 20mL de diluente, com inseminação intra-uterina, obteve-se um bom desempenho reprodutivo.