Extraction of DNA from Sperm Cells in Mixed Stain by Nylon Membrane Bushing Separation Technique / 法医学杂志

OBJECTIVES@#To establish a novel method for the separation of sperm cells in mixed stain, and to evaluate its application value.@*METHODS@#Totally 40 mixed stain samples were collected from sexual assault cases. Sperm cells were separated by the conventional differential lysis method and the nylon membrane bushing separation technique, respectively. The DNA of sperm cells was extracted with the silicon membrane kit （Forensic DNA Extraction Kit for Soft Tissues）. The PCR amplification was performed using AmpFℓSTR® Identifiler® Plus kit, and the products were electrophoresed by 3500xL genetic analyser. The results of two separation methods were then compared.@*RESULTS@#Complete and single-source male STR genotypes could be obtained from all the 40 mixed stain samples except three samples with minimal residual of female DNA by the nylon membrane bushing separation technique. The STR genotypes of sperm cells could not be detected in 25 samples, which were obtained in 15 samples （seven were of incomplete male STR genotypes, six with residual of female DNA, two were complete and single-source STR genotypes of sperm cells）.@*CONCLUSIONS@#The nylon membrane bushing separation technique developed in present study can be used in the separation of sperm cells in mixed stain, especially for the extraction of a small amount of sperm from a large quantity of female cells, which is inexpensive, rapid and simple.

Application value of frozen-thawed mouse round spermatids in in vitro fertilization / 中华男科学杂志

Objective@#To investigate the application value of the frozen-thawed round spermatids of the mouse in in vitro fertilization (IVF).@*METHODS@#Haploid spermatids of the mouse obtained in vitro were divided into a frozen-thawed and a fresh group and oocytes were collected from 6－8 weeks old female mice. After diamidino-phenyl-indole (DAPI) staining, the oocytes were subjected to intracytoplasmic round spermatid injection (ROSI), 259 in the frozen-thawed and 238 in the fresh group. Comparisons were made between the two groups in the capacities of fertilization and embryonic development.@*RESULTS@#The survival rate of the frozen-thawed haploid spermatids was (75.9 ± 2.3) %. No statistically significant differences were observed between the frozen-thawed and fresh groups after ROSI in the rates of fertilization (51.9 vs 55.7%, P >0.05), 2-cell embryos (51.0 vs 62.2%, P >0.05), 4－8-cell embryos (41.8 vs 42.9%, P >0.05), or morula-blastocysts (12.2 vs 21.4%, P >0.05).@*CONCLUSIONS@#Frozen-thawed round spermatids of the mouse are similar to fresh ones in their capacities of fertilization and embryonic development.

Multipotent male germline stem cells (mGSCs) from neonate porcine testis

ABSTRACT Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis, during which unlimited spermatozoa is produced daily derived from SSCs in the testis throughout life of the male. Germline stem (GS) cells can be isolated from spermatogonia, which shared the characteristics of SSCs and embryonic stem cells (ESCs), and can be passaged stably in vitro. The study of GS cells contributes to understanding spermatogenesis process. However, little is known about the GS cells in domestic animals. Here, we report the successful establishment of a serum- and feeder-free system for multipotent male GS cells (mGSCs) from postnatal porcine testis. These cells expressed pluripotent markers, such as Oct4, Nanog, C-myc, and germline-specific markers including Vasa, CD90, CD49f, Gfrα1, Plzf and Dazl. Then we assayed the developmental potential of these cells in vitro. The porcine multipotent male germline stem cells (pmGSCs) can form embryoid bodies (EBs) by suspension culture. Immunofluorescence analysis showed that the EBs differentiated into neuron-specific enolase (NSE, ectoderm), α-actin (mesoderm), and Pdx1 (endoderm) positive cells. These cells induced by 10-6 M retinoic acid (RA) could be differentiated into spermatid-like cells which were positive for Acrosin. The pmGSCs has been cultured over 14 passages. Thus, we have established a long-term culture system for pmGSCs. This culture system provides a platform for the study of porcine mGSCs.

Histological Alterations in the Rabbit Epididymis Induced by Sildenafil Overdosing.

Background: Sildenafil is used for the treatment of erectile dysfunction and is helping millions of men around the world to achieve and maintain a long lasting erection. The histological alterations in the genital system epithelial lining due to sildenafil overdoses intoxication has not yet been well documented. Aim: The present study was carried out to investigate the histological alterations induced by sildenafil overdoses in the epididymal epithelial lining. Methodology: Fifty adult male rabbits (Oryctolagus cuniculus) were subjected to sildenafil (0, 1, 3, 6, 9 mg/kg/day) for 5 days per week for 7 weeks. Samples from left and right proximal epididymis were applied to conventional histological techniques and subjected to histological examination. Results: Exposure to overdoses of sildenafil had provoked histological alterations in the epididymal tissue in the form of epididymal hyperplasia and dilated lumina. Mature spermatozoa were less frequent in the lumen of the epididymis than the control ones while spermatogenic cells, cellular debris and spermatid giant cell were seen in the lumen of the epididymis. Conclusion: The results of the present study confirms that sildenafil provoked alterations in the epididymal epithelial lining together with partial arresting spermatogenesis and impairing the spermatic cells differentiation towards maturation. The findings together might indicate an affect on male fertility induced by sildenafil overdoses.

Intracytoplasmic spermatid injection and in vitro maturation: fact or fiction?

Intracytoplasmic injection with testicular spermatozoa has become a routine treatment in fertility clinics. Spermatozoa can be recovered in half of patients with nonobstructive azoospermia. The use of immature germ cells for intracytoplasmic injection has been proposed for cases in which no spermatozoa can be retrieved. However, there are low pregnancy rates following intracytoplasmic injection using round spermatids from men with no elongated spermatids or spermatozoa in their testes. The in vitro culture of immature germ cells to more mature stages has been proposed as a means to improve this poor outcome. Several years after the introduction of intracytoplasmic injection with elongating and round spermatids, uncertainty remains as to whether this approach can be considered a safe treatment option. This review outlines the clinical and scientific data regarding intracytoplasmic injection using immature germ cells and in vitro matured germ cells.

Spermiogenesis and spermatozoa ultrastructure in the Serrasalminae (Ostariophysi: Characiformes) with further evidence on the relationships of the piranhas and pacus

Spermatozoa and spermiogenesis ultrastructure were studied in the serrasalmine species Piaractus mesopotamicus, Mylossoma duriventre, Serrasalmus maculatus, and Metynnis mola and two distinct patterns may be recognized: the first common to Mylossoma, Serrasalmus and Metynnis, and the other, characteristic of Piaractus. The latter pattern is more similar to the conditions found in Salminus and Brycon. On the other hand, serrasalmine spermatozoa also share characteristics with the spermatozoa of species of the superfamily Anostomoidea. The phylogenetic significance of these characters is discussed.(AU)

A ultraestrutura dos espermatozoides e espermiogênese foram estudadas nas espécies Piaractus mesopotamicus, Mylossoma duriventre, Serrasalmus maculatus e Metynnis mola de Serrasalminae, sendo dois padrões distintos reconhecidos: um comum a Mylossoma, Serrasalmus e Metynnis, e o segundo característico de Piaractus. Este último é mais similar ao observado em Salminus e Brycon. Por outro lado, o espermatozoide dos serrasalmíneos compartilha algumas características com espécies da superfamília Anostomoidea. O significado filogenético destes caracteres é discutido.(AU)

Comparative analysis of spermiogenesis and sperm ultrastructure in Callichthyidae (Teleostei: Ostariophysi: Siluriformes)

In Corydoradinae, the presence of spermatids in the lumen of the testicular tubules together with spermatozoa suggests that spermatogenesis is of the semicystic type, whereas in Callichthyinae, sperm production occurs entirely within spermatocysts in the germinal epithelium, characterizing cystic spermatogenesis. Spermiogenesis in Callichthyinae is characterized by an initial lateral development of the flagellum, the presence of nuclear rotation to different degrees, an eccentric or medial formation of a nuclear fossa, formation of a cytoplasmic channel, and presence of centriolar migration, being more similar to type I spermiogenesis. In Corydoradinae, spermiogenesis is characterized by eccentric development of the flagellum, the absence of nuclear rotation, an eccentric nuclear fossa formation, formation of a cytoplasmic channel, and absence of centriolar migration, differing from the types previously described. The process of spermatogenesis and spermiogenesis in Corydoradinae and Callichthyinae revealed unique characters for each of these subfamilies, corroborating the hypotheses that they constitute monophyletic groups. In relation to sperm ultrastructure, the comparative analysis of the callichthyid species shows that the general characteristics found in the spermatozoa were similar, thus, reinforcing the hypothesis that the family is monophyletic

Em Corydoradinae, a presença de espermátides junto com espermatozóides no lúmen dos túbulos testiculares sugere uma espermatogênese do tipo semicística, enquanto que em Callichthyinae a produção do esperma ocorre inteiramente dentro dos espermatocistos no epitélio germinativo, caracterizando a espermatogênese cística. A espermiogênese em Callichthyinae é caracterizada por um desenvolvimento inicial lateral do flagelo, pela presença de rotação nuclear em diferentes graus, formação de uma fossa nuclear excêntrica ou medial, formação de um canal citoplasmático, e presença de migração centriolar, sendo mais similar à espermiogênese do tipo I. Em Corydoradinae, a espermiogênese é caracterizada pelo desenvolvimento excêntrico do flagelo, ausência de rotação nuclear, fossa nuclear excêntrica, formação de um canal citoplasmático, e ausência de migração centriolar, diferindo dos tipos descritos previamente. O processo de espermatogênese e espermiogênese em Corydoradinae e Callichthyinae revelaram caracteres únicos para cada subfamília, corroborando a hipótese de que as mesmas constituem grupos monofiléticos. Em relação à ultraestrutura do esperma, a análise comparativa das espécies de Callichthyidae mostra que as características gerais encontradas nos espermatozóides foram similares, reforçando a hipótese de monofilia da família

IMMUNOFLUORESCENCE ANALYSIS OF MANCHETTE DURING MOUSE SPERMIOGENESIS / 解剖学报

Objective To investigate the localization and the morphological changes of manchette during mouse spermiogenesis.Methods Immunofluorescence staining with FITC and costaining with DAPI were used to demonstrate the cellular localization of the manchette at different stages during mouse spermiogenesis.The structural changes of the manchette were observed during the maturing of the spermatid.Results Immunofluorescence staining showed that manchette existed exactly around the nuclei of the spermatids.Manchette began to form,when the shape of the nucleus changed from spherical to slightly elongated.While the nucleus of the spermatids condensed and elongated at later stages,manchette moved gradually to the caudal position of the spermatids.At last,the manchette diminished as the spermatids became mature.During mouse spermiogenesis,manchette underwent a transition from a cap-like to a tubular configuration.ConclusionThe formation and diminishment of the manchette is in step with the condensation and elongation of the nucleus of the spermatid.Both the structural and positional changes of the manchette coincide with the changes of the nucleus.These results imply that manchette might play an important role in mouse spermiogenesis.

HISTOCHEMICAL OBSERVATION OF NUCLEOPROTEIN TRANSITION IN SPERMATOGENIC CELLS OF RAT TESTIS / 解剖学报

During spermatogenesis, lysine-rich histones in spermatogenic cells are progressively replaced by arginine-rich protamine. In this study, the nucleoprotein transition in spermatogenic cells of Wistar rat was investigated using aniline blue staining, PTA staininig (both to demonstrate lysine-proteins) and NQS (1, 2-naphthoquinone4-sodium sulfonate) staining (to demonstrate arginine-protein). Under light microscope, the nuclei of spermatogonia and spermatocytes were intensely aniline blue positive and the nuclei of young spermatids moderately positive, while the nuclei of late elongated spermatids and spermatozoa were aniline blue negative. The nuclei of spermatogonia and young spermatids were basicallyy NQS negative and there were a few granules of weak NQS positive in the nuclei of primary spermatocytes, while the nuclei of late elongated spermatids and spermatozoa were NQS positive. Under the electron microscope, the PTA positive chromatin in the nuclei of early spermatids with round nucleus (steps 1 to 8)was fibrillar or granular in appearance. Along with the condensation of nuclei of spermatids(steps 9 to 13), the nuclear stain ability increased. The positive chromatin in the nuclei of spermatids (steps 14, 15) was disappeared progressively, in the direction from cranial to caudal along with the further condensation of nuclei. The nuclei of late spermatids with elongated nucleus (steps 18, 19) and spermatozoa were PTA negative. These observations suggest that the nucleoprotein transition from histones to protamine (S_1) occur during spermiogenesis and that this process could be divided into two consecutive steps, i. e. from histones through an intermediate phase of transition proteins to protamine.