Platelet-rich plasma (PRP) containing autologous growth factors is applied in regenerative medicine, but the lack of an optimized PRP preparation protocol causes unstable therapeutic effects. The aim of this study was to optimize the PRP preparation method and compare the effects of PRP from different preparation methods in restoration of erectile function in a rat model. The in vivo experiments used Sprague–Dawley male rats (n = 24), which were randomly divided into four groups of equal numbers: group I underwent sham operation, while the remaining three groups underwent bilateral CN crush. Crush injury groups were treated at the time of injury with an application of general PRP, optimized PRP [with the largest amount of platelet-derived growth factor (PDGF)–AB] or normal saline-only injection in the corpus cavernosum, respectively. Four weeks later, erectile function was assessed by CN electrosimulation, and penile tissue was collected for histology. Results demonstrated that in the PRP group prepared with the ACD-A anticoagulant, chitosan and incubated at −20°C for 15 days had the largest amount of PDGF-AB and showed a synergistic effect on release (p < 0.05). Functional outcome measurement and immunofluorescence staining for the dorsal nerve revealed that improvement after bilateral CN injury occurred in the optimized PRP group (p < 0.05). It was concluded that optimized PRP with a high level of growth factors was more stable, and its injection into the corpus cavernosum facilitated recovery of erectile function. Copyright © 2013 John Wiley & Sons, Ltd.

The d-galactose (d-gal)-injected animal model, which is typically established by administering consecutive subcutaneous d-gal injections to animals for approximately six or eight weeks, has been frequently used for aging research. In addition, this animal model has been demonstrated to accelerate aging in the brain, kidneys, liver and blood cells. However, studies on aging in male reproductive organs that have used this animal model remain few. Therefore, the current study aimed to optimize a model of male reproductive aging by administering d-gal injections to male mice and to determine the possible mechanism expediting senescence processes during spermatogenesis. In this study, C57Bl/6 mice were randomized into five groups (each containing 8–10 mice according to the daily intraperitoneal injection of vehicle control or 100 or 200 mg/kg dosages of d-gal for a period of six or eight weeks). First, mice subjected to d-gal injections for six or eight weeks demonstrated considerably decreased superoxide dismutase activity in the serum and testis lysates compared to those in the control group. The lipid peroxidation in testis also increased in the d-gal-injected groups. Furthermore, the d-gal-injected groups exhibited a decreased ratio of testis weight/body weight and sperm count compared to the control group. The percentages of both immotile sperm and abnormal sperm increased considerably in the d-gal-injected groups compared to those of the control group. To determine the genes influenced by the d-gal injection during murine spermatogenesis, a c-DNA microarray was conducted to compare testicular RNA samples between the treated groups and the control group. The d-gal-injected groups exhibited RNA transcripts of nine spermatogenesis-related genes (Cycl2, Hk1, Pltp, Utp3, Cabyr, Zpbp2, Speer2, Csnka2ip and Katnb1) that were up- or down-regulated by at least two-fold compared to the control group. Several of these genes are critical for forming sperm-head morphologies or maintaining nuclear integration (e.g., cylicin, basic protein of sperm head cytoskeleton 2 (Cylc2), casein kinase 2, alpha prime interacting protein (Csnka2ip) and katanin p80 (WD40-containing) subunit B1 (Katnb1)). These results indicate that d-gal-injected mice are suitable for investigating male reproductive aging.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF) and are associated with congenital bilateral absence of the vas deferens (CBAVD), which is the major cause of infertility in male patients with CF. However, most Taiwanese patients with CBAVD do not carry major CFTR mutations. Some patients have a single copy deletion of the solute carrier family 9 isoform 3 (SLC9A3) gene. SLC9A3 is a Na⁺/H⁺ exchanger, and depleted Slc9a3 in male mice causes infertility due to the abnormal dilated lumen of the rete testis and efferent ductules. Furthermore, SLC9A3 interacts with CFTR in the pancreatic duct and functions as a genetic modifier of CF. However, SLC9A3 function and its relation to CFTR expression in the male reproductive tract in vivo remain elusive. In the present study, we found that CFTR expression was dramatically decreased in the epididymis and vas deferens of Slc9a3 knockout mice. Adult Slc9a3^-/- mice showed not only significantly decreased epididymis and vas deferens weight but also increased testis weight. Furthermore, Slc9a3^-/- mice developed obstructive azoospermia because of abnormal abundant secretions and calcification in the lumen of the reproductive tract. Ultrastructural analysis of the epithelium in Slc9a3^–/–epididymis and vas deferens displayed disorganized and reduced number of stereocilia and numerous secretory apparatuses. Our data revealed that interdependence between SLC9A3 and CFTR is critical for maintaining a precise microenvironment in the epithelial cytoarchitecture of the male reproductive tract. The Slc9a3-deficient mice with impaired male excurrent ducts in this study provide proof for our clinical findings that some Taiwanese of CBAVD carry SLC9A3 deletion but without major CFTR mutations. Cystic fibrosis (CF) is the most common inherited life-threatening disease in Caucasians. The most well-known cause of CF is a genetic defect in CFTR, an apical membrane chloride and bicarbonate channel. The symptoms of CF include defects in the respiratory, digestive, and male reproductive systems. Most male patients with CF are infertile due to congenital bilateral absence of the vas deferens (CBAVD), which leads to obstructive azoospermia. Nevertheless, Taiwanese patients with CBAVD do not carry the common mutations of CFTR found in Caucasians. We have identified a potential candidate, SLC9A3, of which a single copy is lost in Taiwanese patients with CBAVD. In addition to the previously reported role of SLC9A3 in the digestive system and efferent ductules, we now report that the SLC9A3 deficiency causes obstructive azoospermia and impairs the epithelial structure of the reproductive tract. Loss of SLC9A3 also leads to dramatic reduced expression of CFTR in the reproductive tract. We suggest that the interplay between SLC9A3 and CFTR is responsible for CF-related infertility. Thus, we have characterized a potential critical player in the pathogenesis of CBAVD and provide a new diagnostic candidate for Asian patients with CBAVD.

According to recent estimates, 2%–15% of couples are sterile, and approximately half of the infertility cases are attributed to male reproductive factors. However, the reasons remain undefined in approximately 25% of male infertility cases, and most infertility cases exhibit spermatogenic defects. Numerous genes involved in spermatogenesis still remain unknown. We previously identified Male Germ Cells Rab GTPase-Activating Proteins (MGCRABGAPs) through cDNA microarray analysis of human testicular tissues with spermatogenic defects. MGCRABGAP contains a conserved RABGAP catalytic domain, TBC (Tre2/Bub2/Cdc16). RABGAP family proteins regulate cellular function (e.g., cytoskeletal remodeling, vesicular trafficking, and cell migration) by inactivating RAB proteins. MGCRABGAP is a male germ cell-specific protein expressed in elongating and elongated spermatids during mammalian spermiogenesis. The purpose of this study was to identify proteins that interact with MGCRABGAP during mammalian spermiogenesis using a proteomic approach. We found that MGCRABGAP exhibited GTPase-activating bioability, and several MGCRABGAP interactors, possible substrates (e.g., RAB10, RAB5C, and RAP1), were identified using co-immunoprecipitation (co-IP) and nano liquid chromatography-mass spectrometry/mass spectrometry (nano LC-MS/MS). We confirmed the binding ability between RAB10 and MGCRABGAP via co-IP. Additionally, MGCRABGAP–RAB10 complexes were specifically colocalized in the manchette structure, a critical structure for the formation of spermatid heads, and were slightly expressed at the midpiece of mature spermatozoa. Based on these results, we propose that MGCRABGAP is involved in mammalian spermiogenesis by modulating RAB10.

Congenital bilateral absence of the vas deferens (CBAVD) is a distinct clinical entity accounting for ∼25% of obstructive azoospermia in infertile men. The association between CBAVD and mutated CFTR (cystic fibrosis transmembrane conductance regulator) alleles is well demonstrated in Caucasians, but the identity of CBAVD-susceptibility genes remains elusive in Asians. We investigate genomic copy number variations (CNVs) in a patient cohort of Taiwan. Genome-wide screening for genetic CNVs was conducted on eight individuals with CBAVD using array-based comparative genomic hybridization. One recurrent CNV was detected on 3q26.1 in five patients, and another was detected on a reproduction-related gene PANK2 in two patients. For the former, we further characterized the breakpoints in CBAVD and assessed the incidence in healthy individuals by tiling path arrays. The deletion in each patient was confirmed, and seven out of the eight controls were also affected. Examination of the homozygous loss of PANK2 by PCR in a larger cohort showed a homozygous deletion in only one of the 26 CBAVD males, and not in any of the 20 azoospermic patients without CBAVD, nor in any of the 16 control subjects. Our results suggest that 3q26.1 may not be a critical region for CBAVD. Additionally no strong association was found for PANK2 in this reproduction disorder. Other reproduction-related genes, such as PBX1, BRD3, COL18A1 and HMOX1, identified by this initial study may inspire further investigation.