Effects of chemotherapeutic agent bendamustine for non-hodgkin lymphoma on spermatogenesis in mice.

Non-Hodgkin lymphoma (NHL) is one of the most common cancers affecting men of reproductive age. The high response rate of bendamustine as first-line treatment for NHL, coupled with young age of patients, makes elucidation of the impact of treatment on male reproduction important. Our aim was to determine the effects of bendamustine on male reproduction by animal model. Male mice were treated with bendamustine (40 mg/kg) through tail vein injection while cisplatin was given as a standard (3 mg/kg) through intraperitoneal injection. After 3 weeks, bendamustine induced weight loss and sperm morphology abnormalities were compared to the control. Additionally, sperm with folded tails were the most frequent abnormality in bendamustine-treated mice. But the mechanism of sperm abnormality induced by bendamustine remains to be elucidated. These results indicate bendamustine may affect spermatozoa of patients who have been treated for NHL.

Different effects of androgen on the expression of Fut1, Fut2, Fut4 and Fut9 in male mouse reproductive tract.

The α-(1,2) fucosyltransferases (Fut1 and Fut2) and α-(1,3) fucosyltransferases (Fut4, Fut9) are responsible for the synthesis of Lewis X (LeX) and Lewis Y (LeY) conjugated to glycoproteins. We recently reported that these fucosyltransferases were differentially expressed in the reproductive tract of male mouse. Here, we studied the effect of androgen on fucosyltransferase expression through the use of mouse castration models. We found that Fut1 mRNA and Fut4 mRNA were upregulated, while Fut2 mRNA and Fut9 mRNA were downregulated by androgen in the caput epididymis. However, in the vas deferens and prostate, only Fut4 mRNA and Fut2 mRNA were respectively upregulated following exposure to androgen. In the seminal vesicle, all fucosyltransferases, with the exception of Fut9, were upregulated. We identified the androgen receptor binding sites (ARBSs) of Fut2, Fut4 and Fut9 in the caput epididymis. Luciferase assay for these ARBSs is able to provide an indication as to why Fut4 and Fut9 are differently expressed and regulated by androgen, although they catalyze the same α-(1,3) fucose linkage. Our study showed that androgen could differentially regulate the expression of these fucosyltransferases and provided an insight into the characteristic distribution of each fucosyltransferase responsible for LeX/LeY biosynthesis in the male reproductive tract.

Bioinformatics for spermatogenesis: annotation of male reproduction based on proteomics.

Proteomics strategies have been widely used in the field of male reproduction, both in basic and clinical research. Bioinformatics methods are indispensable in proteomics-based studies and are used for data presentation, database construction and functional annotation. In the present review, we focus on the functional annotation of gene lists obtained through qualitative or quantitative methods, summarizing the common and male reproduction specialized proteomics databases. We introduce several integrated tools used to find the hidden biological significance from the data obtained. We further describe in detail the information on male reproduction derived from Gene Ontology analyses, pathway analyses and biomedical analyses. We provide an overview of bioinformatics annotations in spermatogenesis, from gene function to biological function and from biological function to clinical application. On the basis of recently published proteomics studies and associated data, we show that bioinformatics methods help us to discover drug targets for sperm motility and to scan for cancer-testis genes. In addition, we summarize the online resources relevant to male reproduction research for the exploration of the regulation of spermatogenesis.

Integrity of the blood-testis barrier in healthy men after suppression of spermatogenesis with testosterone and levonorgestrel.

STUDY QUESTION: Do exogenous male hormonal contraceptives that suppress intratesticular testosterone and spermatogenesis interfere with the blood-testis barrier integrity in men? SUMMARY ANSWER: When spermatogenesis was suppressed by testosterone alone or combined with levonorgestrel (LNG) treatment in men, the structural appearance of Sertoli cell tight junctions remained intact in the human testis. WHAT IS ALREADY KNOWN: Testosterone promotes the integrity of the blood-testis barrier. Intratesticular androgen deprivation induced by exogenous testosterone plus a progestin to suppress spermatogenesis in a contraceptive regimen may disturb the structural and functional integrity of the blood-testis barrier. STUDY DESIGN, SIZE AND DURATION: Testicular biopsies were obtained from a sub-study of a randomized clinical trial of 36 healthy Chinese men who were treated for 18 weeks and followed for at least a 12-week recovery period. PARTICIPANTS/MATERIAL, SETTING, METHODS: Healthy Chinese male volunteers (27-48 years) were randomized to two treatment groups (n = 18/group) for 18 weeks: (1) testosterone undecanoate (TU) 1000 mg i.m. injection followed by a 500 mg injection every 6 weeks and (2) TU + LNG 250 µg orally daily. Blood samples were obtained from all participants before and during treatment and at the end of the recovery phase. Open testicular biopsies for this study were obtained from four men before treatment and from four men in each of the TU and TU + LNG groups at 2 and 9 weeks of treatment. The presence of antisperm antibodies was checked in the archived serum samples of the subjects at baseline, during treatment and at the end of the recovery period. Stored testicular biopsy samples from cynomolgus monkeys treated with either sub-cutaneous testosterone or placebo for 12 weeks were used for additional protein expression studies. MAIN RESULTS AND ROLE OF THE CHANCE: Expression of blood-testis barrier associated proteins quantified by immunohistochemistry (claudin 3, claudin 11, junctional adhesion molecule-A, zonula occludens-1) remained unchanged despite a significant decrease in the numbers of pachytene spermatocytes and round spermatids in the seminiferous tubules at 9 weeks in the TU + LNG group. This was confirmed by immunoblots showing a lack of quantitative change in these tight junction proteins in monkeys after testosterone treatment. There were no increases in serum antisperm antibodies in the volunteers during the study. LIMITATIONS/REASONS FOR CAUTION: The duration of the study was short and the long-term effects of male hormonal contraceptive treatments on the integrity of the blood-testis barrier remain to be determined. WIDER IMPLICATIONS OF THE FINDINGS: This study supports the safety of male hormonal contraceptive treatment and does not corroborate the previous findings of disturbed immunological integrity of the blood-testis barrier from animal studies such as androgen receptor knockout mice and exogenous hormonal treatment in rats. STUDY FUNDING/COMPETING INTEREST: The study was supported by grants from the Contraceptive Research and Development Program and the Mellon Foundation (MFG-02-64, MFG-03-67), Endocrine, Metabolism and Nutrition Training Grant (T32 DK007571), the Clinical and Translational Science Institute at Los Angeles Biomedical and Harbor-UCLA Medical Center (UL1RR033176 and UL1TR000124) and the Los Angeles Biomedical Research Institute Summer High School Student Program.

[Location of Dctn1 in the mouse testis and sperm and its role in spermiogenesis].

OBJECTIVE: To investigate the role of dynactin 1 (Dctn1) in the process of mouse spermiogenesis. METHODS: Western blot and indirect immunofluorescence were used to analyze the expression and location of Dctn1 in the mouse testis and spermatozoa. The highest efficiency of small interference RNA (siRNA) was verified by GC2-spd cell line in vitro and in vivo studies, respectively. Dctn1 siRNA mixed with the indicator (0.4% trypan blue) was injected into the seminiferous tubules of 3-week-old ICR mice through rete testis microinjection, and negative control siRNA injected into the control testes. The normal group included 3-week-old ICR mice that did not receive any treatment. Spermatozoa were collected from the cauda epididymis 3 weeks after siRNA injection for morphological analysis. RESULTS: Dctn1 was mainly localized in the tail of spermatozoa. After interference, the sperm tail abnormality in the Dctn1 siRNA group was (23.57 +/- 0.55)%, significantly higher than (12.35 +/- 2.29)% in the control (P < 0.01, n = 3), and it was (3.37 +/- 0.69)% in the normal group. CONCLUSION: Dctn1 plays an important role in mouse spermiogenesis, and mainly affects the formation of the tail of spermatozoa.

Effects of di-n-butyl phthalate on male rat reproduction following pubertal exposure.

Di-n-butyl phthalate (DBP) is an endocrine-disrupting chemical that has the potential to affect male reproduction. However, the reproductive effects of low-dose DBP are still not well known, especially at the molecular level. In the present study, pubertal male Sprague-Dawley rats were orally administered DBP at a wide range of doses (0.1, 1.0, 10, 100 and 500 mg kg⁻¹ day⁻¹) for 30 days. The selected end points included reproductive organ weights, testicular histopathology and serum hormonal levels. Additionally, proteomic analysis was performed to identify proteins that are differentially expressed as a result of exposure to DBP at low doses (0.1, 1.0 and 10 mg kg⁻¹ day⁻¹). Toxic effects were observed in the high-dose groups, including anomalous development of testes and epididymides, severe atrophy of seminiferous tubules, loss of spermatogenesis and abnormal levels of serum hormones. Treatment with low doses of DBP seemed to exert a 'stimulative effect' on the serum hormones. Proteomics analysis of rat testes showed 20 differentially expressed proteins. Among these proteins, alterations in the expression of HnRNPA2/B1, vimentin and superoxide dismutase 1 (SOD1) were further confirmed by Western blot and immunohistochemistry. Taken together, we conclude that high doses of DBP led to testicular toxicity, and low doses of DBP led to changes in the expression of proteins involved in spermatogenesis as well as changes in the number and function of Sertoli and Leydig cells, although no obvious morphological changes appeared. The identification of these differentially expressed proteins provides important information about the mechanisms underlying the effects of DBP on male rat reproduction.

[Expression of annexin A7 in spermatogonial stem cells].

OBJECTIVE: To study the expression of Annexin A7 in the mouse testis, especially in different types of spermatogonia. METHODS: We prepared Annexin A7 recombinant protein using prokaryotic expression, adsorbed the Annexin A7 antibody with it after identified by mass spectrometry, and detected the expression of Annexin A7 by Western-blot and immunohistochemistry. RESULTS: Annexin A7 was expressed in a development-dependent manner in the spermatogonia of the prepubertal mice and in the type-A single (As) and type-A paired (Apr) spermatogonia of adult mice. These results were confirmed by the co-localization of Annexin A7 and Stra8, a known determinant of differentiated spermatogonial stem cells (SSCs). CONCLUSION: Annexin A7 is the internal factor of As and Apr spermatogonia, which might be involved in the biological functions of SSCs.

The role of ezrin-associated protein network in human sperm capacitation.

Membrane modifications in sperm cells represent a key step in sperm capacitation; however, the molecular basis of these modifications is not fully understood. Ezrin is the best-studied member of the ezrin/radixin/merlin family. As a cross-linker between the cortical cytoskeleton and plasma membrane proteins, ezrin contributes to remodeling of the membrane surface structure. Furthermore, activated ezrin and the Rho dissociation inhibitor, RhoGDI, promote the formation of cortical cytoskeleton-polymerized actin through Rho activation. Thus, ezrin, actin, RhoGDI, Rho and plasma membrane proteins form a complicated network in vivo, which contributes to the assembly of the structure of the membrane surface. Previously, we showed that ezrin and RhoGDI1 are expressed in human testes. Thus, we sought to determine whether the ezrin-RhoGDI1-actin-membrane protein network has a role in human sperm capacitation. Our results by Western blot indicate that ezrin is activated by phosphorylation of the threonine567 residue during capacitation. Co-immunoprecipitation studies revealed that, during sperm capacitation, the interaction between ezrin and RhoGDI1 increases, and phosphostaining of two dimensional electrophoresis gels showed that RhoGDI1 is phosphorylated, suggesting that RhoGDI1 dissociates from RhoA and leads to actin polymerization on the sperm head. We speculate that activated ezrin interacts with polymerized actin and the glycosylated membrane protein cd44 after capacitation. Blocking sperm capacitation using ezrin- or actin-specific monoclonal antibodies decreases their acrosome reaction (AR) rate, but has no effect on the AR alone. Taken together, our results show that a network consisting of ezrin, RhoGDI1, RhoA, F-actin and membrane proteins functions to influence the modifications that occur on the membrane of the sperm head during human sperm capacitation.

[46, XX testicular disorder of sex development: report of 2 cases and review of the literature].

OBJECTIVE: To investigate the phenotype, pathogenesis and molecular biological features of 46, XX testicular disorder of sex development. METHODS: We obtained the history of 2 patients with 46, XX testicular disorder of sex development, examined the cavitas pelvis by type-B ultrasonography, analyzed the karyotype of the chromosome, and detected the genes SRY, YRRM1, DYS240 and DAZ by PCR amplification. RESULTS: Microrchidia, azoospermia and maldevelopment of secondary sex characteristics were observed in both of the patients, but ultrasonography revealed no female internal genitals. Their chromosome gender was karyotyped as 46, XX, with the SRY gene positive in both, but the YRRM1 gene positive in only one of the cases. CONCLUSION: Chromosome karyotyping and detection of the SRY gene for patients with abnormal sex development can give us an insight into the genetic pathogenesis and provide us with scientific evidence for the diagnosis and treatment of the condition.

AF-2364 is a prospective spermicide candidate.

AbstractInhibition of sperm motility has recently become a promising target for male contraceptive development. AF-2364, an analogue of Lonidamine (LND), had a contraceptive effect when orally administered to adult Sprague-Dawley rats. LND can also target mitochondria to inhibit oxygen consumption and block energy metabolism in tumour cells. However, there are no reports of the effects of AF-2364 on human sperm function. Herein we describe the action of AF-2364 on human sperm in vitro, as well as the mechanisms involved. AF-2364 specifically blocked human sperm motility in vitro. Further experiments revealed that AF-2364 can target sperm mitochondrial permeability transition (MPT) pores to induce the loss of sperm mitochondrial membrane potential (DeltaPsim) and decrease ATP generation; however, no significant changes in the cytoskeletal network or the human sperm proteome were detected after exposure to AF-2364. Incubation of AF-2364 with other human or mouse cell lines indicated that the spermicidal effect at the lower concentration was specific. In summary, the spermicidal effect of AF-2364 involves direct action on sperm MPT pores, and this compound should be further investigated as a new spermicide candidate.

[Expression of carbonic anhydrase II in human testes and spermatozoa and its clinical significance].

OBJECTIVE: To investigate the expression of carbonic anhydrase II (CA2) in human testes and spermatozoa, and to compare the expressions of CA2 in ejaculated spermatozoa between normozoospermic and asthenozoospermic men. METHODS: The localization of CA2 in human testes was observed by immunohistochemistry, and that in human sperm by immunofluorescence. Western blot was used to detect the expression of CA2 in the semen samples obtained from 16 normozoospermic and 16 asthenozoospermic volunteers. RESULTS: The CA2 protein was shown to be localized in the tail of elongating spermatids by immunohistochemistry and in the flagellum of human sperm by immunofluorescence. Western blot revealed an obviously increased expression of CA2 in the spermatozoa of asthenozoospermic patients, with statistically significant difference from the normozoospermic group (1.84 +/- 0.32 vs 1.41 +/- 0.26, P < 0.05). CONCLUSION: The CA2 protein is expressed in the spermatogenic stage of elongating spermatids in human testes and localized in the sperm tail. The expression of CA2 is significantly increased in the spermatozoa of asthenozoospermic men, which might be responsible for low sperm motility.

The proteomic analysis of human neonatal umbilical cord serum by mass spectrometry.

AIM: To investigate the proteome composition and function of human neonatal arterial umbilical cord. METHODS: Serum proteomic analyses were performed on samples from both males and females by using a combination of techniques: (1) removal of six high-abundance proteins, (2) tryptic digestion of low-abundance proteins, (3) separation of peptide mixtures by reverse-phase high-performance liquid chromatography (RP-HPLC), and (4) peptide identification using electrospray ionization tandem mass spectrometry (ESI-MS/MS). RESULTS: A total of 837 non-redundant proteins were identified, with 213 male-specific and 239 female-specific proteins. Among them, 319 proteins were identified by at least 2 distinct peptides. The subcellular localization, function, and pathway involvement for each of the identified proteins were analyzed. A comparison of this neonatal proteome to that of adult serum proteome revealed novel biomarkers, such as alpha-fetoprotein and periostin that were specific to newborn infants. CONCLUSION: These data will contribute to a better understanding of the composition of umbilical cord serum and aid the discovery of novel biomarkers for the prenatal diagnosis of fetal abnormalities.

NYD-SP27, a novel intrinsic decapacitation factor in sperm.

Prior to fertilization sperm has to undergo an activation process known as capaciation, leading to the acrosome reaction. Till now, little is known about the mechanism for preventing premature capacitation in sperm although decapacitation factors from various sources have been thought to be involved. In this study, we report that NYD-SP27, an isoform of phospholipase C Zeta 1 (PLCZ1), is localized to the sperm acrosome in mouse and human spermatozoa by immunofluorescence using a specific antibody. Western blot and double staining analyses show NYD-SP27 becomes detached from sperm, as they undergo capacitation and acrosome reaction. The absence of HCO3-, a key factor in activating capacitation, from the capacitation-inducing medium prevents the loss of NYD-SP27 from sperm. The anti-NYD-SP27 antibody also prevents the loss of NYD-SP27 from sperm, reduced the number of capacitated sperm, inhibited the acrosome reaction induced by ATP and progesterone, and inhibited agonist-induced PLC-coupled Ca2+ mobilization in sperm, which can be mimicked by the PLC inhibitor, U73122. These data strongly suggest that NYD-SP27 is a physiological inhibitor of PLC that acts as an intrinsic decapacitation factor in sperm to prevent premature capacitation and acrosome reaction.

Role of translation by mitochondrial-type ribosomes during sperm capacitation: an analysis based on a proteomic approach.

Mammalian spermatozoa contain a complex population of mRNAs, some of which have been demonstrated to be translated de novo by mitochondrial-type ribosomes using D-chloramphenicol (CP), a specific inhibitor of mitochondrial translation. However, little is known about the functions of these mRNAs in mature sperm. In the present study, differential proteomic approaches were applied to study sperm protein profiles translated by mitochondrial-type ribosomes using the inhibitor CP and 44 proteins were identified with lower expression in CP-treated sperm in comparison to capacitated sperm (ratio >or= 1.5, p<0.05). Results of Western blot and real-time PCR suggest that four proteins were translated by mitochondrial-type ribosomes. Bioinformatics analysis indicated that 26 of 44 proteins were involved in some critical processes correlated to sperm-egg interaction event. In addition, Mups, whose functions in reproduction have never been studied, were chosen for further study. Our results showed that Mups proteins were localized to the acrosome and flagellum of precapacitated sperm, and were also expressed in the equatorial segment of capacitated sperm. The depletion of Mups using neutralizing antibodies significantly inhibited capacitation in a dose-dependent manner, subsequently inhibited acrosome reaction and sperm-egg fusion. In summary, mitochondrial translation during capacitation can store proteins beneficial for sperm-egg interaction.

Characterization of Spindlin1 isoform2 in mouse testis.

AIM: To investigate the expression of Spindlin 1 (Spin 1) isoform2 and assess its function in mouse testis. METHODS: First, reverse-transcription polymerase chain reaction (RT-PCR) was used to determine whether Spin1 isoform2 is present in mouse testis. Then the expression patterns of the isoform between newborn and adult mice testes were compared by immunoblot analysis. Finally, the diversity of its localization in mice testes at different ages (days 0, 7, 14, 21, 28 and 60) was observed by immunohistochemistry. The localization of the protein in mouse sperm was also investigated by immunofluorescence. RESULTS: The RT-PCR results show that Spin1 isoform2 is present in mouse testis. As shown by immunoblot analysis, the isoform was more highly expressed in adult testes compared with newborn testes. Interestingly, Spin1 isoform2 did not show up in the cytoplasm of primary spermatocytes until day 14. Also, the protein exists at the tail of the mouse sperm. CONCLUSION: Spin1 isoform2 is a protein expressed highly in adult testis, which might be involved in spermatogenesis and could be necessary for normal sperm motility.

Construction of a proteome profile and functional analysis of the proteins involved in the initiation of mouse spermatogenesis.

Spermatogenesis is a complex process of terminal differentiation wherein mature sperm are produced. In the first wave of mouse spermatogenesis, different spermatogenic cells appear at specific time points, and their appearance is expected to be accompanied by changes in specific protein expression patterns. In this study, we used 2D-PAGE and MALDI-TOF/TOF technology to construct a comparative proteome profile for mouse testis at specific time points (days 0, 7, 14, 21, 28, and 60 postpartum). We identified 362 differential protein spots corresponding to 257 different proteins. Further cluster analysis revealed 6 expression patterns, and bioinformatics analysis revealed that each pattern was related to many specific cell processes. Among them, 28 novel proteins with unknown functions neither in somatic cells nor germ cells were identified, 8 of which were found to be uniquely or highly expressed in mouse testes via comparison with the GNF SymAtlas database. Further, we randomly selected 7 protein spots and the above 8 novel proteins to verify the expression pattern via Western blotting and RT-PCR, and 6 proteins with little information in testis were further investigated to explore their cellular localization during spermatogenesis by performing immunohistochemistry for the mouse testis tissue. Taken together, the above results reveal an important proteome profile that is functional during the first wave of mouse spermatogenesis, and they provide a strong basis for further research.

transient scrotal hyperthermia and levonorgestrel enhance testosterone-induced spermatogenesis suppression in men through increased germ cell apoptosis.

CONTEXT: In rodents and monkeys, a combination of hormonal and physical agents accelerates germ cell death. OBJECTIVE: A "proof of concept" study was performed to investigate whether addition of heat exposure or a progestin to an androgen induces germ cell death and more complete and rapid spermatogenesis suppression. DESIGN AND SETTINGS: A randomized clinical trial was performed at academic medical centers. PARTICIPANTS: We treated four groups of healthy male volunteers (18 per group) for 18 wk: 1) testosterone undecanoate (TU) 1000 mg im (first dose), followed by 500 mg im every 6 wk; 2) submersion of scrota at 43 C in water for 30 min/d for 6 consecutive days; 3) TU plus heat; and 4) TU plus oral levonorgestrel (LNG) 250 microg/d. MAIN OUTCOME MEASURES: Semen parameters, testicular histology, and germ cell apoptosis were the main outcome measures. RESULTS: Heat alone and TU plus heat suppressed sperm counts more than TU alone by wk 6. By wk 9, recovery began in the heat only group, whereas spermatogenesis remained suppressed in the TU plus heat group. Oral LNG plus TU suppressed spermatogenesis earlier and more severely than TU alone. At wk 2, significantly greater germ cell apoptosis occurred in heat and heat plus TU subjects, but not in subjects without heat treatment, compared with pretreatment subjects. By 9 wk, markedly smaller seminiferous tubule diameters and fewer spermatocytes and spermatids were noted in all 12 biopsies from men receiving TU, TU plus LNG, with most dramatic differences for the TU plus heat group, whereas no differences from pretreatment biopsies were observed in men who received heat treatment only. CONCLUSIONS: Heat causes a rapid and transient suppression of spermatogenesis. TU plus heat resulted in low-sperm output that was maintained by continuous treatment with TU. Addition of an oral progestin accelerated spermatogenesis suppression by TU alone. Increased germ cell apoptosis contributed to suppression of spermatogenesis.

[Update of the researches on sperm proteome].

Proteomics and polemic techniques are among the most valuable approaches in the research of life science in this new century, marking the beginning of a post-genome era. Two-dimensional electrophoresis and mass spectrometry, applied as key techniques in proteomic research, have given rise to new research strategies and improved the efficiency of researchers in exploring the unknown field. Its introduction into the exploration of spermatozoal proteins has given us so many pleasant surprises. This review presents some essential information about proteomics and two-dimensional electrophoresis and mass spectrometry, with a brief introduction of the recent progress in the researches on human sperm proteome, capacitation-related sperm proteins, sperm-egg interaction-related proteins, and sperm-immunity which has made great senses in the clinical problems.

[Effects of Jujingwan on nitric oxide and superoxide dismutase in seminal plasma of asthenospermia patients].

OBJECTIVE: To explore the possible action mechanism of Jujingwan on asthenospermia. METHODS: Semen routine analyses and determination of nitric oxide (NO) concentration and superoxide dismutase (SOD) activity in seminal plasma were performed in 34 cases of asthenospermia. The changes of NO concentration and SOD activity before and after the treatment with Jujingwan were observed. RESULTS: Compared with pre-treatment, there was no significant change in NO concentration, and the activity of SOD decreased significantly after the treatment ([95.97 +/- 20.75] microg/L vs [6.14 +/- 19.99] microg/L). There was negative correlation between NO concentration and SOD activity before the treatment (r = -0.246, P < 0.05). CONCLUSION: Jujingwan can significantly improve sperm viability in patients with asthenospermia. However, the excellent effects of Jujingwan are not displayed in the changes of NO concentration and SOD activity.