Dwi A. Pujianto

Regional Expression of Androgen Receptor Coregulators and Androgen Action in the Mouse Epididymis

Endocrine regulation of the mouse initial segment (IS) and distal caput epididymides was studied using genome-wide profiling of gene expression. Among the IS-enriched genes, 29% were significantly down-regulated 1 day after gonadectomy. Of those genes, dihydrotestosterone (DHT) supplementation was not sufficient to maintain their pregonadectomy level of expression in 70%. Of the caput-enriched genes, 16% were significantly down-regulated after gonadectomy, and of those genes, DHT supplementation did not maintain the initial level of expression in 28%. Identical data were obtained by clustering analyses performed for the expression data of epididymal genes. Furthermore, the microarray data revealed that 26 androgen receptor coregulators were expressed in the epididymis, of which several were confirmed by quantitative reverse transcriptase polymerase chain reaction analysis. This suggests putative involvement of these proteins in the segment-specific regulation of the epididymal genes. The pattern of epididymal gene expression in the novel proximal epididymis-specific androgen receptor knockout mouse ProxE-ARKO, with severe hypotrophy and hypoplasia of the caput epithelium, furthermore suggested that a subset of genes whose expression cannot be maintained by systemic androgen alone still require either direct lumicrine androgen action or a permissive effect of circulating testosterone. It is evident that testicular factors, one of which could be the high-concentration luminal androgen, are important for the expression of IS-enriched genes, whereas the expression of distal caput-enriched genes is typically regulated by systemic androgens.

Sperm-associated antigen 11A is expressed exclusively in the principal cells of the mouse caput epididymis in an androgen-dependent manner.

BackgroundEpididymal sperm maturation occurs via interactions between sperm and proteins secreted by the epididymal epithelium. Although this is an important process, the genes that encode the involved proteins remain largely uncharacterized. Previous studies have demonstrated that the genes involved in sperm maturation are regulated by androgen. Spag11a is an epididymal gene that is influenced by androgen. However, little is known about the putative role of this gene in the sperm maturation process. The objective of this study was to characterize Spag11a in the mouse epididymis.MethodsIn silico analyses were performed to predict signal peptides and functional domains. Spag11a expression was measured by quantitative real-time RT-PCR. Western blots and immunocytochemistry were performed to determine protein expression.ResultsSPAG11A is a member of the beta defensin protein family and constitutes a secretory protein. Spag11a was expressed exclusively in the epididymis. Moreover, it exhibited region-specific expression in the caput, which is typical for genes that are involved in creating a suitable microenvironment for sperm maturation. Mouse Spag11a was regulated by androgen. A significant decrease of Spag11a expression was observed at third day following a gonadectomy (P < 0.001). Interestingly, testosterone replacement therapy was able to maintain the expression almost at the normal level, indicating a dependency on androgen. Besides androgen, testicular factors influenced Spag11a expression in a different way. This was revealed by efferent duct ligation in which Spag11a was transiently up-regulated at the third day following the ligation before returning to the normal level at day 5. Spag11a regional expression was also observed at protein level detected by western immunoblotting which revealed a clear band in the caput but not in other regions. The prediction that SPAG11A is a secretory protein was confirmed by immunocytochemical analyses indicating cell-specific expression mainly in the caput principal cells and detection of the protein in epididymal luminal fluid and spermatozoa.ConclusionsBased on the characteristics of Spag11a, it is likely that this gene has a specific role in epididymal sperm maturation. Further studies using functional assays are necessary to confirm this finding.

Sperm Na+, K+-ATPase α4 and plasma membrane Ca2+-ATPase (PMCA) 4 regulation in asthenozoospermia

ABSTRACT Asthenozoospermia, which is characterized by reduced motility, is one of the etiologies of male infertility. Its biochemical and functional consequences include altered ATPase activity. This study investigated the activities of Na+, K+-ATPase and Ca2+-ATPase and the expression of Na+, K+-ATPase α4 and PMCA4 isoforms in human sperm of asthenozoospermic infertile men. Nineteen samples from asthenozoospermic infertile couples were examined in this study. Computerized-assisted semen analysis (CASA) was performed, and the enzyme activity was measured based on the ability of ATPase to release organic phosphate from ATP as a substrate. The Na+, K+-ATPase α4 and PMCA4 isoform expression levels were measured by western immunoblotting, whereas the protein distribution was examined by immunocytochemistry. This showed that the Na+, K+-ATPase activity and the Na+, K+-ATPase α4 isoform expression were lower in the asthenozoospermia group than in the normozoospermia group (8.688±1.161 versus 13.851±1.884 µmol Pi/mg protein/h, respectively; p>0.05). In contrast, the Ca2+-ATPase activity was significantly higher in the asthenozoospermia group than in the normozoospermia group (11.154±1.186 versus 2.725±0.545 µmol Pi/mg protein/h, respectively; p<0.05). In comparison, PMCA4 expression in the asthenozoospermia group was lower than in the normozoospermia group (p>0.05). The altered ATPase activity and isoform expression in asthenozoospermia may impair sperm structure and function.

Sperm quality after swim up and density gradient centrifugation sperm preparation with supplementation of alpha lipoic acid (ALA): A preliminary study

Intra uterine insemination (IUI) as one of the treatment for infertility, persists low success rate. A factor that contributes to the unsuccessful of IUI is sperm preparation, performed through Swim-up (SU) and Density Gradient Centrifugation (DGC) methods. Furthermore, studies have shown that Alpha Lipoic Acid (ALA) is a potent antioxidant that could enhance the sperm motility and protect the DNA integrity of the sperm [1]. This study is aimed to re-evaluate the efficiency of the DGC and SU methods in selecting sperm before being transferred for IUI by the supplementation of ALA based on the sperm DNA integrity. Semen samples were obtained from 13 men from partners of women who are infertile (normozoospermia) and underwent IUI. Semen analysis based on the guideline of World Health Organization (WHO) 2010 was performed to measure the sperm motility and velocity, before and after sperm preparation. Then, samples were incubated with Alpha Lipoic Acid (ALA) in 0.625 mg (ALA 1), 1.25 mg (ALA 2) and 2.5 mg (ALA 3). The Sperm Chromatin Dispersion (SCD) test was performed to evaluate the sperm DNA Fragmentation Index (DFI). The percentage of motile sperm was higher in prepared sperm (post-DGC and post-SU) than in whole semen. Furthermore, the percentage of motile sperm was higher in post-DGC compared to post-SU. The level of DFI after the supplementation of ALA was decreased in prepared sperm compared to the whole semen. ALA was proved capable to select the better sperm quality with decreased sperm DNA fragmentation of prepared sperm in the all of DFI category.Intra uterine insemination (IUI) as one of the treatment for infertility, persists low success rate. A factor that contributes to the unsuccessful of IUI is sperm preparation, performed through Swim-up (SU) and Density Gradient Centrifugation (DGC) methods. Furthermore, studies have shown that Alpha Lipoic Acid (ALA) is a potent antioxidant that could enhance the sperm motility and protect the DNA integrity of the sperm [1]. This study is aimed to re-evaluate the efficiency of the DGC and SU methods in selecting sperm before being transferred for IUI by the supplementation of ALA based on the sperm DNA integrity. Semen samples were obtained from 13 men from partners of women who are infertile (normozoospermia) and underwent IUI. Semen analysis based on the guideline of World Health Organization (WHO) 2010 was performed to measure the sperm motility and velocity, before and after sperm preparation. Then, samples were incubated with Alpha Lipoic Acid (ALA) in 0.625 mg (ALA 1), 1.25 mg (ALA 2) and 2.5 mg (AL...

Anti-VDAC3 recombinant antibody decreased human sperm motility and membrane integrity: A potential spermicide for contraception

Objective: To express recombinant protein that comprises an important fragment of human sperm specific voltage dependent anion channel 3 (VDAC3) protein as a potential molecule for generation of antibody, which can affect sperm function, aiming at spermicide development. Methods: The produce of VDAC3 recombinant protein encoded by cDNA sequence of human VDAC3 exon 5-8, based on experimental design of VDAC3 knock-out mice study. And after the purification of various human sperm VDAC3 recombinant proteins, epitope has been predicted in our recombinant protein determined by ElliPro program. Polyclonal antibody was produced for 14 wk. Then anti-VDAC3-exon 5-8 recombinant antiserum was inoculated to human sperm. After the process, antibody VDAC3 protein in human sperm was incubation with anti-VDAC3 recombinant antibody. Finally evaluation the effect of VDAC3 antiserum to human sperm motility and plasma membrane integrity was proceeded. Results: Human VDAC3 recombinant protein was successfully over-expressed in Escherichia coli and purified by affinity chromatography method. Purified human sperm VDAC3 recombinant protein could stimulate immune response in rabbit producing an antibody against VDAC3. Anti-VDAC3 recombinant antibody recognized VDAC3 antigen in human sperm could decrease human sperm motility and membrane integrity significantly. Conclusions: Anti-VDAC3 recombinant polyclonal antibody that we produced in rabbit by ourselves could decrease sperm motility and sperm membrane integrity. The authors suggest this polyclonal antibody could be used as a candidate agent for male contraception in the future. Furthermore, the authors intend to explore the effect of this antibody into sperm function aiming at male contraceptive vaccine development.

The Effect of Injection with Papaya (Carica Papaya L.) Seed Extract on Sperm Concentration and Spermatogenic Cells of Male Rats (Rattus norvegicus L.) Strain LMR.

So far men as a subject in family planning program had no priority, however recently men become a focus. Established mothodes for male contraception are through condom and vasectomy. Using condoms create psychological complaints, whereas vasectomy although very effective has often permanent effect. An other method of contraception is hormonal; besides that it is important to develop contraception using plants with antifertility effect such as papaya seed. Therefore, the aim of this research is to know the effect of extract papaya seeds on concentration and viability of sperms in vas deferens of male rat Strain LMR. This research was done using papaya seed extract, Bangka variety with 7 treatments, doses/kg/body weight, including 0 mg; 0.1 mg; 0.5 mg; 0.9 mg; 1.0 mg; 5.0 mg; 9.0 mg for times each treatment. Administration of papaya seed extract was performed by intramusculary injection for 20 days (1,5 seminiferous epithelium cycles). Investigation were done on 1) sperms concentration of vas deferens, 2) weight of testis, 3) seminiferous tubules diametric, 4) condition of spermatogenic cells. Injection with papaya seed extract for 20 days increased sperm concentration of vas deferens significantly (Pl0,01), decreased population of spermatogonium A and primary spermatocytes preleptoten significantly (pl0,05), did not give any significant effect on weight of testis, seminiferous tubules diametric, primary spermatocytes pachyten and spermatid (Pg0,05)