Search

Microplastic pollution is an emerging stressor of concern to coral reef ecosystems, which are already threatened by additional global and local level anthropogenic stressors. The effects of ingesting microplastics alone on corals have been well studied, but the effects of the chemical composition of these particles have been understudied. Many microplastic-associated chemicals are endocrine disrupters potentially posing a threat to organismal reproduction. Therefore, the goal of this study was to determine if differences exist between the effects of microplastics themselves and microplastic leachate on Montipora capitata fertilization due to changes in fatty acid quantity and composition. Assays were conducted two years in a row which exposed M. capitata gamete bundles to either one of four types of recently manufactured, virgin microspheres (nylon, polypropylene, high-density polyethylene, or low-density polyethylene) at three concentrations (50, 100, or 200 particles/L) or microplastic leachates, presumably including plastic additives from these microspheres. Gamete fertilization was not impacted by microplastic particles themselves, but some of the microplastic leachate treatments with the same polymer type significantly reduced fertilization rates for M. capitata. Additionally, a total of 17 fatty acids were seen in both years, but neither fatty acid quantity nor composition correlated with observed declines in fertilization. Instead, fertilization and fatty acid data independently varied by concentration and polymer type, likely due to the presence of different chemicals. This study is the first to directly test the toxicity of microplastic leachate to coral reproduction. These findings show that microplastic-associated chemicals are an important stressor affecting successful coral fertilization and fatty acid quantity and composition and provide evidence for the negative effects of microplastic leachate to coral reproduction. Thus, plastic additives could pose an additional threat to coral replenishment and persistence in coral reef ecosystems., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Primordial germ cells (PGCs) are the founding population of the germ cell lineage that undergo a multistep process to generate spermatozoa or oocytes. Establishing an appropriate culture system for PGCs is a key challenge in reproductive biology. By a chemical screening using mouse PGC-like cells (mPGCLCs), which were induced from mouse embryonic stem cells, we reported previously that forskolin and rolipram synergistically enhanced the proliferation/survival of mPGCLCs with an average expansion rate of ~20-fold. In the present study, we evaluated other chemicals or cytokines to see whether they would improve the current mPGCLC culture system. Among the chemicals and cytokines examined, in the presence of forskolin and rolipram, cyclosporin A (CsA) and fibroblast growth factors (FGFs: FGF2 and FGF10) effectively enhanced the expansion of mPGCLCs in vitro (~50-fold on average). During the expansion by CsA or FGFs, mPGCLCs comprehensively erased their DNA methylation to acquire a profile equivalent to that of gonadal germ cells in vivo, while maintaining their highly motile phenotype as well as their transcriptional properties as sexually uncommitted PGCs. Importantly, these mPGCLCs robustly contributed to spermatogenesis and produced fertile offspring. Furthermore, mouse PGCs (mPGCs) cultured with CsA ex vivo showed transcriptomes and DNA methylomes similar to those of cultured mPGCLCs. The improved culture system for mPGCLCs/mPGCs would be instructive for addressing key questions in PGC biology, including the mechanisms for germ cell migration, epigenetic reprogramming, and sex determination of the germline., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Study Question: Does experimental manipulation of fibroblast growth factor 9 (FGF9)-signalling in human fetal gonads alter sex-specific gonadal differentiation?, Summary Answer: Inhibition of FGFR signalling following SU5402 treatment impaired germ cell survival in both sexes and severely altered the developing somatic niche in testes, while stimulation of FGF9 signalling promoted Sertoli cell proliferation in testes and inhibited meiotic entry of germ cells in ovaries., What Is Known Already: Sex-specific differentiation of bipotential gonads involves a complex signalling cascade that includes a combination of factors promoting either testicular or ovarian differentiation and inhibition of the opposing pathway. In mice, FGF9/FGFR2 signalling has been shown to promote testicular differentiation and antagonize the female developmental pathway through inhibition of WNT4., Study Design, Size, Duration: FGF signalling was manipulated in human fetal gonads in an established ex vivo culture model by treatments with recombinant FGF9 (25 ng/ml) and the tyrosine kinase inhibitor SU5402 (10 μM) that was used to inhibit FGFR signalling. Human fetal testis and ovary tissues were cultured for 14 days and effects on gonadal development and expression of cell lineage markers were determined., Participants/materials, Setting, Methods: Gonadal tissues from 44 male and 33 female embryos/fetuses from first trimester were used for ex vivo culture experiments. Tissues were analyzed by evaluation of histology and immunohistochemical analysis of markers for germ cells, somatic cells, proliferation and apoptosis. Culture media were collected throughout the experimental period and production of steroid hormone metabolites was analyzed in media from fetal testis cultures by liquid chromatography-tandem mass spectrometry (LC-MS/MS)., Main Results and the Role of Chance: Treatment with SU5402 resulted in near complete loss of gonocytes (224 vs. 14 OCT4+ cells per mm2, P < 0.05) and oogonia (1456 vs. 28 OCT4+ cells per mm2, P < 0.001) in human fetal testes and ovaries, respectively. This was a result of both increased apoptosis and reduced proliferation in the germ cells. Addition of exogenous FGF9 to the culture media resulted in a reduced number of germ cells entering meiosis in fetal ovaries (102 vs. 60 γH2AX+ germ cells per mm2, P < 0.05), while in fetal testes FGF9 stimulation resulted in an increased number of Sertoli cells (2503 vs. 3872 SOX9+ cells per mm2, P < 0.05). In fetal testes, inhibition of FGFR signalling by SU5402 treatment altered seminiferous cord morphology and reduced the AMH expression as well as the number of SOX9-positive Sertoli cells (2503 vs. 1561 SOX9+ cells per mm2, P < 0.05). In interstitial cells, reduced expression of COUP-TFII and increased expression of CYP11A1 and CYP17A1 in fetal Leydig cells was observed, although there were no subsequent changes in steroidogenesis., Large Scale Data: N/A., Limitations, Reasons for Caution: Ex vivo culture may not replicate all aspects of fetal gonadal development and function in vivo. Although the effects of FGF9 were studied in ex vivo culture experiments, there is no direct evidence that FGF9 acts in vivo during human fetal gonadogenesis. The FGFR inhibitor (SU5402) used in this study is not specific to FGFR2 but inhibits all FGF receptors and off-target effects on unrelated tyrosine kinases should be considered., Wider Implications of the Findings: The findings of this study suggest that dysregulation of FGFR-mediated signalling may affect both testicular and ovarian development, in particular impacting the fetal germ cell populations in both sexes., Study Funding/competing Interest(s): This work was supported in part by an ESPE Research Fellowship, sponsored by Novo Nordisk A/S to A.JØ. Additional funding was obtained from the Erichsen Family Fund (A.JØ.), the Aase and Ejnar Danielsens Fund (A.JØ.), the Danish Government's support for the EDMaRC programme (A.JU.) and a Wellcome Trust Intermediate Clinical Fellowship (R.T.M., Grant no. 098522). The Medical Research Council (MRC) Centre for Reproductive Health (R.T.M.) is supported by an MRC Centre Grant (MR/N022556/1). The authors have no conflict of interest to disclose., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)

Diabetes is associated with poor oocyte quality and the dysregulation of ovarian function and is thus a leading contributor to the increasing prevalence of female reproductive pathologies. Accordingly, it is well-established that insulin fulfills a key role in the regulation of several facets of female reproduction. What remains less certain is whether proinsulin C-peptide, which has recently been implicated in cellular signaling cascades, holds a functional role in the female germline. In the present study, we examined the expression of insulin, C-peptide, and its purported receptor; GPR146, within the mouse ovary and oocyte. Our data establish the presence of abundant C-peptide within follicular fluid and raise the prospect that this bioactive peptide is internalized by oocytes in a G-protein coupled receptor-dependent manner. Further, our data reveal that internalized C-peptide undergoes pronounced subcellular relocalization from the ooplasm to the pronuclei postfertilization. The application of immunoprecipitation analysis and mass spectrometry identified breast cancer type 2 susceptibility protein (BRCA2), the meiotic resumption/DNA repair protein, as a primary binding partner for C-peptide within the oocyte. Collectively, these findings establish a novel accumulation profile for C-peptide in the female germline and provide the first evidence for an interaction between C-peptide and BRCA2. This interaction is particularly intriguing when considering the propensity for oocytes from diabetic women to experience aberrant meiotic resumption and perturbation of traditional DNA repair processes. This therefore provides a clear imperative for further investigation of the implications of dysregulated C-peptide production in these individuals., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Humans are universally exposed to low levels of phthalate esters (phthalates), which are used to plasticize polyvinyl chloride. Phthalates exert adverse effects on the development of seminiferous cords in the fetal testis through unknown toxicity pathways. To investigate the hypothesis that phthalates alter seminiferous cord development by disrupting retinoic acid (RA) signaling in the fetal testis, gestational day 15 fetal rat testes were exposed for 1-3 days to 10-6 M all-trans retinoic acid (ATRA) alone or in combination with 10-6-10-4 M mono-(2-ethylhexyl) phthalate (MEHP) in ex vivo culture. As previously reported, exogenous ATRA reduced seminiferous cord number. This effect was attenuated in a concentration-dependent fashion by MEHP co-exposure. ATRA and MEHP-exposed testes were depleted of DDX4-positive germ cells but not Sertoli cells. MEHP alone enhanced the expression of the RA receptor target Rbp1 and the ovary development-associated genes Wnt4 and Nr0b1, and suppressed expression of the Leydig cell marker, Star, and the germ cell markers, Ddx4 and Pou5f1. In co-exposures, MEHP predominantly enhanced the gene expression effects of ATRA, but the Wnt4 and Nr0b1 concentration-responses were nonlinear. Similarly, ATRA increased the number of cells expressing the granulosa cell marker FOXL2 in testis cultures, but this induction was attenuated by addition of MEHP. These results indicate that MEHP can both enhance and inhibit actions of ATRA during fetal testis development and provide evidence that RA signaling is a target for phthalate toxicity in the fetal testis., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Many of the testicular cancer-survived patients, treated with chemotherapeutic drugs, show infertility, pre and postimplantation loss, and germ cell abnormality. Studies examining the negative effects of chemotherapeutic drugs on testicular germ cells are ongoing; however, information on the stemness properties and proteomic profiles of these cells are lacking. This study investigated the effects of chemotherapeutic drugs etoposide, cisplatin, bleomycin, and their combination (BEP) on the physiology and stem cell activity of mouse germ cells in vitro. Our results showed that treatment with the abovementioned drugs affected germ cell viability and decreased the number of proliferating germ cells significantly at specific concentrations (0.05 µM etoposide, 1 µM cisplatin, 10 µM bleomycin, and 0.1 µM BEP), which maintained a survival rate of >90%. We also observed a significantly higher percentage of apoptotic cells and alterations in the expression of undifferentiated and differentiated spermatogonia-related genes and marker proteins in germ cells exposed to abovementioned concentrations of the drugs. Next, we performed germ cell transplantation into recipient mice and observed a remarkable reduction in stemness properties of spermatogonial stem cells at these concentrations. Based on these results, we assessed the levels of differentially expressed proteins by performing proteomic analysis. We found that treatment with the abovementioned drugs induced cell damage, oxidative stress, metabolic disruption, and immune deficiency which may promote tumor regeneration, cytotoxicity, infertility, and transgenerational cellular function transmission. Thus, this study provides information about the chemotherapy-induced recurrent destruction and thereby can lead possible changes in medication.

Ganciclovir (GCV) has been implicated in the development of testicular alterations. Exposure on gestational day (GD) 10 in rats induced permanent effects, including focal reduction or absence of germ cells (Sertoli cell-only tubules). Because the timing of exposure can be critical for testicular effects, we exposed rat dams to 300 mg/kg GCV (3 100 mg/kg subcutaneous injections) on GD10, 14 and 19, when germ cells have high rates of migration, proliferation and are mitotically quiescent, respectively. Males exposed to GCV in utero on GD10 and 14 were evaluated for androgenization markers, serum and fecal androgens, and testicular histomorphometry at adulthood. Double-labeling immunofluorescence for DAZL and Ki67 were used to assess gonocytes number and the proliferative activity of germ and somatic cells in fetal testes on GD15 and 20, ie, 24 h after GCV exposure. Adult rats exposed on GD14 showed delayed puberty onset, despite normal androgen levels. Also, there was a 50% reduction in testicular weight and about 30% of seminiferous tubules lacking germ cells. Effects on GD10 animals were less pronounced. In the fetal testis, the number of gonocytes was reduced by 50% in rats exposed on GD14, but normal in GD19 fetuses. GCV also reduced Sertoli cell proliferation immunolabeling in GD19 fetuses and Sertoli cell number in adults. In conclusion, GCV toxicity on germ cells seems to be linked to their proliferation rate and GD14 is a critical window in rats, when GCV exposure causes an acute massive loss of germ cells that persists until adulthood.

Endocrine-disrupting chemicals (EDCs) generate reproductive dysfunctions affecting the biosynthesis of steroid hormones and genes of the steroidogenic pathway. EDCs effects are mainly reported as a result of exposure to single compounds. However, humans are environmentally exposed to a mixture of EDCs. Herein, we assess chronic exposure to single alkylphenols and phthalates versus a mixture in mouse testes histology and steroidogenesis. Pregnant mice were exposed through drinking water to: 0.3 mg/kg-body weight (BW)/d of each phthalate (bis (2-ethylhexyl) phthalate, dibutyl phthalate, benzyl butyl phthalate), 0.05 mg/kg-BW/d of each alkylphenol (4-nonylphenol, 4-tert-octylphenol), or their mixture, covering from 0.5 postcoital day to weaning, continuing in the male offspring each exposure until adulthood (60-days old). Body and relative testis weight were increased in mixture-exposed mice along with histological alterations. Intratesticular testosterone (T) changed only in mice exposed to DBP, whereas estradiol (E2) levels were altered in all groups (except benzyl butyl phthalate). mRNA levels of genes encoding hormones of the steroid pathway (Cyp11a1, Hsd3b1, Cyp17a1, and Cyp19a1), cholesterol transporters (Star), and transcriptional factors (Sp1) showed that mice exposed to single or mixed compounds had alterations in at least 2 transcripts. However, none of the different types of exposure induced changes in all transcripts. In addition, changes at the mRNA or protein levels with single compounds were not always the same as those with a mixture. In conclusion, the effects of a chronic exposure to a mixture of EDCs on the expression of genes and proteins of the steroidogenic pathway and hormonal status were different from those exposed to single EDC., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Single low-dose primaquine (PQ) reduces Plasmodium falciparum infectivity before it impacts gametocyte density. Here, we examined the effect of PQ on gametocyte sex ratio as a possible explanation for this early sterilizing effect., Methods: Quantitative reverse-transcription polymerase chain reaction assays were developed to quantify female gametocytes (targeting Pfs25 messenger RNA [mRNA]) and male gametocytes (targeting Pf3D7&#95;1469900 mRNA) in 2 randomized trials in Kenya and Mali, comparing dihydroartemisinin-piperaquine (DP) alone to DP with PQ. Gametocyte sex ratio was examined in relation to time since treatment and infectivity to mosquitoes., Results: In Kenya, the median proportion of male gametocytes was 0.33 at baseline. Seven days after treatment, gametocyte density was significantly reduced in the DP-PQ arm relative to the DP arm (females: 0.05% [interquartile range {IQR}, 0.0-0.7%] of baseline; males: 3.4% [IQR, 0.4%-32.9%] of baseline; P < .001). Twenty-four hours after treatment, gametocyte sex ratio became male-biased and was not significantly different between the DP and DP-PQ groups. In Mali, there was no significant difference in sex ratio between the DP and DP-PQ groups (>0.125 mg/kg) 48 hours after treatment, and gametocyte sex ratio was not associated with mosquito infection rates., Conclusions: The early sterilizing effects of PQ may not be explained by the preferential clearance of male gametocytes and may be due to an effect on gametocyte fitness., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.)

Polybrominated diphenyl ethers may affect male reproductive function; however, the underlying mechanism is still uncertain. By using Caenorhabditis elegans, a commonly used model to study basic biological processes in apoptosis, we investigated the toxic effects of 4-bromodiphenyl ether (BDE-3), the most fundamental mono-BDE generated from degradation of polybrominated diphenyl ethers in the environment. We found that BDE-3 treated worms exhibited decreased life spans, impaired fecundity and delayed egg laying. BDE-3 induced dose-dependent germ cell apoptosis in wild-type N2 strain; however, this effect was blocked in mutants of p53/cep-1 and DNA damage response gene hus-1. Moreover, the knockout of the MAPK kinases (mutants mek-1 and sek-1) and the p53 antagonist protein ABL-1 (abl-1), which are essential for stress-induced germ cell apoptosis, also abrogated the germ cell apoptosis induced by BDE-3. Generation of reactive oxygen species (ROS) in intact animals was determined by a fluorescent probe, 2,7-dichlorofluorescein diacetate, and the ROS level was significantly elevated by BDE-3 treatment. Microarray analysis on gene expression profiles further revealed the possible pathways involved in BDE-3 toxicity. Overall, our findings suggested that BDE-3 could induce reproductive dysfunction and germ cell apoptosis in C. elegans by induction of ROS and DNA damage., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Effects of ZnO NPs and ionic Zn on germline apoptosis and the regulation of genes in the apoptosis pathway were investigated in vivo using the model organism Caenorhabditis elegans.Age synchronized Bristol N2 worms were exposed to ZnO NPs and ZnCl2 at concentrations of 6.14 × 10-1, 61.4, and 614 μM form larval stage 1 (L1) to early adulthood. Possible ZnO nanoparticles were observed under the worm cuticle and also in the gonadal region by transmission electron microscopy (TEM). ZnO NPs and ZnCl2 both significantly increased the number of apoptotic cells as compared with controls in the 61.4 and 614 μM treatment groups (P < .05). However, ZnO NPs induced more apoptotic cells in the 61.4 μM treatment than ZnCl2 (P <  .05), suggesting ZnO NP is more potent in inducing apoptosis at specific exposure concentration. Findings using the MD701 (bcIs39 [(lim-7)ced-1p::GFP + lin-15(+)]) strain further confirmed the observations in N2 strain. Genes involved in the apoptosis pathway (ced-13, ced-3, ced-4, ced-9, cep-1, dpl-1, efl-1, efl-2, egl-1, egl-38, lin-35, pax-2, and sir-2.1) were in general upregulated in response to ZnO NP exposure. The cep-1/p53 gene was up-regulated in gene expression assay. In the cep-1 loss of function mutant, no significant increase in apoptosis was observed. Therefore, the increased apoptosis resulting from ZnO NPs exposure is likely cep-1/p53 dependent. This study provides evidence that ZnO nanoparticles affect germ cell apoptotic machinery as a potential mechanism of reproductive toxicity., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Inflammation can cause degenerative changes of reproductive function. Oleanolic acid (OA), the effective component from Ligustrum lucidum Ait., exhibits significantly anti-inflammation and antiageing activity. However, whether OA restores testicular dysfunction via inhibition of inflammation with ageing is unclear. Here, in a natural ageing rat model, we investigated the protection effects of OA and its mechanism of action., Methods: Eighteen-month-old Sprague Dawley (SD) rats were randomly divided into ageing control group and two OA-treated groups (5 and 25 mg/kg). Nine-month-old SD rats were used as adult controls. All rats were received either vehicle or OA for 6 months. Then, histomorphology, weight and index of testis, protein expression and immunohistochemistry were examined., Key Findings: Oleanolic acid significantly restored testicular morphology and improved testicular weight and index. Moreover, OA significantly inhibited phospho-NF-κB p65 and its downstream proinflammatory cytokines' expressions, including IL-1β, COX-2 and TNF-α in testis tissues. Similarly, OA remarkably inhibited IL-1β and TNF-α production. OA significantly attenuated germ cells' DNA damage and apoptosis. Such changes were accompanied by downregulation of γH2AX, p-P53 and Bax expressions, and upregulation of Bcl-2 and Bcl-2/Bax ratio. In addition, OA remarkably inhibited p38 signalling., Conclusions: Oleanolic acid effectively rejuvenates testicular function via attenuating germ cell DNA damage and apoptosis through deactivation of NF-κB, p53 and p38 signalling pathways., (© 2016 Royal Pharmaceutical Society.)

Humanin is a peptide that is cytoprotective against stresses in many cell types. We investigated whether a potent humanin analogue S14G-humanin (HNG) would protect against chemotherapy-induced damage to normal cells without interfering with the chemotherapy-induced suppression of cancer cells. Young adult male mice were inoculated iv with murine melanoma cells. After 1 week, cancer-bearing mice were randomized to receive either: no treatment, daily ip injection of HNG, a single ip injection of cyclophosphamide (CP), or CP+HNG and killed at the end of 3 weeks. HNG rescued the CP-induced suppression of leucocytes and protected germ cell from CP-induced apoptosis. Lung metastases were suppressed by HNG or CP alone, and further suppressed by CP+HNG treatment. Plasma IGF-1 levels were suppressed by HNG with or without CP treatment. To investigate whether HNG maintains its protective effects on spermatogonial stem cells, sperm output, and peripheral leucocytes after repeated doses of CP, normal adult male mice received: no treatment, daily sc injection of HNG, 6 ip injections of CP at 5-day intervals, and the same regimens of CP+HNG and killed at the end of 4 weeks of treatment. Cauda epididymal sperm counts were elevated by HNG and suppressed by CP. HNG rescued the CP-induced suppression of spermatogonial stem cells, sperm count and peripheral leucocytes. We conclude that HNG 1) protects CP-induced loss of male germ cells and leucocytes, 2) enhances CP-induced suppression of cancer metastases, and 3) acts as a caloric-restriction mimetic by suppressing IGF-1 levels. Our findings suggest that humanin analogues may be promising adjuvants to chemotherapy.

Sperm viability, acrosome integrity, motility, and swimming velocity are determinants of male fertility and exhibit an extreme degree of variation among closely related species. Many of these sperm parameters are associated with sperm ATP content, which has led to predictions of trade-offs between ATP content and sperm motility and velocity. Selective pressures imposed by sperm competition have been proposed as evolutionary causes of this pattern of diversity in sperm traits. Here, we examine variation in sperm viability, acrosome integrity, motility, swimming velocity, and ATP content over time, among 18 species of closely related muroid rodents, to address the following questions: (a) Do sperm from closely related species vary in ATP content after a period of incubation? (b) Are these differences in ATP levels related to differences in other sperm traits? (c) Are differences in ATP content and sperm performance over time explained by the levels of sperm competition in these species? Our results revealed a high degree of interspecific variability in changes in sperm ATP content, acrosome integrity, sperm motility and swimming velocity over time. Additionally, species with high sperm competition levels were able to maintain higher levels of sperm motility and faster sperm swimming velocity when they were incubated under conditions that support sperm survival. Furthermore, we show that the maintenance of such levels of sperm performance is correlated with the ability of sperm to sustain high concentrations of intracellular ATP over time. Thus, sperm competition may have an important role maximizing sperm metabolism and performance and, ultimately, the fertilizing capacity of spermatozoa., (© 2015 by the Society for the Study of Reproduction, Inc.)

Context: Fetal ovarian development and primordial follicle formation underpin future female fertility. Prokineticin (PROK) ligands regulate cell survival, proliferation, and angiogenesis in adult reproductive tissues including the ovary. However, their expression and function during fetal ovarian development remains unclear., Objective: This study aimed to investigate expression and localization of the PROK ligands, receptors, and their downstream transcriptional targets in the human fetal ovary., Setting: This study was conducted at the University of Edinburgh., Participants: Ovaries were collected from 37 morphologically normal human fetuses., Design and Main Outcome Measures: mRNA and protein expression of PROK ligands and receptors was determined in human fetal ovaries using qRT-PCR, immunoblotting, and immunohistochemistry. Functional studies were performed using a human germ cell tumor line (TCam-2) stably transfected with Prokineticin receptor 1 (PROKR1)., Results: Expression of PROK1 and PROKR1 was significantly higher in mid-gestation ovaries (17-20 wk) than at earlier gestations (8-11 and 14-16 wk). PROK2 significantly increased across the gestations examined. PROKR2 expression remained unchanged. PROK ligand and receptor proteins were predominantly localized to germ cells (including oocytes within primordial follicles) and endothelial cells, indicating these cell types to be the targets of PROK signaling in the human fetal ovary. PROK1 treatment of a germ cell line stably expressing PROKR1 resulted in ERK phosphorylation and elevated COX2 expression., Conclusions: Developmental changes in expression and regulation of COX2 and phosphorylated ERK (pERK) by PROK1 suggest that PROK ligands may be novel regulators of germ cell development in the human fetal ovary, interacting within a network of growth and survival factors prior to primordial follicle formation.

Germ cells develop in intimate contact and communication with somatic cells of the gonad. In female mammals, oocyte development depends crucially on gap junctions that couple it to the surrounding somatic granulosa cells of the follicle, yet the mechanisms that regulate this essential intercellular communication remain incompletely understood. Follicle-stimulating hormone (FSH) drives the terminal stage of follicular development. We found that FSH increases the steady-state levels of mRNAs encoding the principal connexins that constitute gap junctions and cadherins that mediate cell attachment. This increase occurs both in granulosa cells, which express the FSH-receptor, and in oocytes, which do not. FSH also increased the number of transzonal projections that provide the sites of granulosa cell-oocyte contact. Consistent with increased connexin expression, FSH increased gap junctional communication between granulosa cells and between the oocyte and granulosa cells, and it accelerated oocyte development. These results demonstrate that FSH regulates communication between the female germ cell and its somatic microenvironment. We propose that FSH-regulated gap junctional communication ensures that differentiation processes occurring in distinct cellular compartments within the follicle are precisely coordinated to ensure production of a fertilizable egg., (© 2015 by the Society for the Study of Reproduction, Inc.)

Endosulfan as a new member of persistent organic pollutants has been shown to induce reproductive dysfunction in various animal models. However, the action mechanism of endosulfan-produced reproductive toxicity remains largely unknown. This study was focused on investigating the reproductive toxicity induced by α-endosulfan and clarifying the role of mitochondria and genotoxic response genes in germ cell apoptosis of Caenorhabditis elegans. Our data showed that endosulfan induced a dose-dependent decrease of life span, fecundity, and hatchability, whereas the germ cell apoptosis was dose-dependently increased. The mitochondria membrane potential was disrupted by endosulfan, leading to a significant increase of germ cell apoptosis in mev-1(kn-1) mutant. However, the apoptotic effects of endosulfan were blocked in mutants of cep-1(w40), egl-1(n487), and hus-1(op241), indicating conserved genotoxic response genes played an essential role in endosulfan-induced germ cell apoptosis. Furthermore, exposure to endosulfan induced the accumulation of HUS-1::GFP foci and the germ cell cycle arrest. These findings provided clear evidence that endosulfan caused significant adverse effects on the reproduction system of C. elegans and increased germ cell apoptosis, which was regulated by mitochondrial dysfunction and DNA damage response genes. This study may help to understand the signal transduction pathways involved in endosulfan-induced reproductive toxicity., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Primordial germ cells (PGCs) can give rise to pluripotent stem cells known as embryonic germ cells (EGCs) when cultured with basic fibroblast growth factor (bFGF), stem cell factor (SCF), and leukemia inhibitory factor. Somatic cells can give rise to induced pluripotent stem cells (iPSCs) by introduction of the reprogramming transcription factors Oct4, Sox2, and Klf4. The effects of Sox2 and Klf4 on somatic cell reprogramming can be reproduced using the small molecule compounds, transforming growth factor-β receptor (TGFβR) inhibitor and Kempaullone, respectively. Here we examined the effects of TGFβR inhibitor and Kempaullone on EGC derivation from PGCs. Treatment of PGCs with TGFβR inhibitor and/or Kempaullone generated pluripotent stem cells under standard embryonic stem cell (ESC) culture conditions without bFGF and SCF, which we termed induced EGCs (iEGCs). The derivation efficiency of iEGCs was dependent on the differentiation stage and sex. DNA methylation levels of imprinted genes in iEGCs were reduced, with the exception of the H19 gene. The promoters of genes involved in germline development were generally hypomethylated in PGCs, but three germline genes showed comparable DNA methylation levels among iEGs, ESCs, and iPSCs. These results show that PGCs can be reprogrammed into pluripotent state using small molecule compounds, and that DNA methylation of these germline genes is not maintained in iEGCs., (© 2014 AlphaMed Press.)

In the present study, we demonstrate that minimal concentrations (≤ 1 nM) of retinoic acid (RA), equivalent to the quantity contaminating serum-containing culture medium, are sufficient to promote meiotic entry and progression through meiotic prophase I (MPI) stages in isolated 12.5-days postcoitum (dpc) XX and XY mouse primordial germ cells (PGCs) in culture. Similarly, we found that the same low RA concentration up-regulated or induced stimulation by retinoic acid 8 (Stra8) in such cells, both at mRNA and protein level. In preleptotene/leptotene germ cells, STRA8 was localized in nuclear dots that disappeared at later MPI stages. In addition to Stra8, other meiotic genes such as Dmc1 and Rec8 appeared stimulated by RA directly in PGCs with similar concentration-dependent trends. Finally, we found that RA induced Stra8, Sycp3, Dmc1, and Rec8 transcripts, promoting meiotic entry in culture also in pregonadal 10.5-dpc PGCs of both sexes. When cultured isolated from somatic cells, such PGCs, however, were unable to progress through MPI stages, while after entering meiosis, they progressed through MPI when cultured within aorta/gonad/mesonephros tissues. We conclude that besides RA, germ cell intrinsic factors and other exogenous signals from the surrounding somatic cells are probably necessary for meiotic entry and progression in mouse PGCs.

Gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25) is a posttranscriptional regulator of genes that are essential for spermatid elongation and completion of spermatogenesis. It also prevents Leydig cells (LCs) from gonadotropin overstimulation of androgen production. In transgenic (Tg) mice carrying deletions of the GRTH 5'-flanking regions, we previously demonstrated that the -1085 bp to ATG contains the elements for basal and androgen-induced LC-specific expression. No expression in germ cells (GCs) was found with sequences extended up to -3.6 kb. To define regulatory regions of GRTH required for expression in GC, Tg mice were generated with 5'-flanking sequence 6.4 kb (6.4 Kb-Tg) and/or deletion using green fluorescent protein (GFP) as reporter gene in the present study. GFP was expressed in all lines. Immunohistochemistry analysis showed that 6.4 Kb-Tg directed GFP expression in both GCs and LCs. Deletion of the sequence -205 bp to -3.6 kb (6.4 Kb/del-Tg) directs GFP expression only in meiotic and haploid GCs. This indicated that the distal region -6.4 kb/-3.6 kb is required for GRTH cell-specific expression in GC. Also, it inhibits the expression of GRTH in LC directed by the 205-bp promoter, an effect that is neutralized by the -3.6-kb/-205-bp sequence. Androgen receptor antagonist, flutamide treatment prevents GFP/GRTH expression in Tg lines, demonstrating in vivo direct and indirect effects of endogenous androgen on LCs and GCs, respectively. Our studies have generated and characterized Tg lines that can be used to define requirements for cell-specific expression of the GRTH gene and to further advance our knowledge on the regulation of GRTH by androgen in GCs.

Sterol-sensing nuclear receptors and insulin-like growth factor signaling play evolutionarily conserved roles in the control of aging. In the nematode Caenorhabditis elegans, bile acid-like steroid hormones known as dafachronic acids (DAs) influence longevity by binding to and regulating the activity of the conserved nuclear receptor DAF-12, and the insulin receptor (InsR) ortholog DAF-2 controls life span by inhibiting the FoxO transcription factor DAF-16. How the DA/DAF-12 pathway interacts with DAF-2/InsR signaling to control life span is poorly understood. Here we specifically investigated the roles of liganded and unliganded DAF-12 in life span control in the context of reduced DAF-2/InsR signaling. In animals with reduced daf-2/InsR activity, mutations that either reduce DA biosynthesis or fully abrogate DAF-12 activity shorten life span, suggesting that liganded DAF-12 promotes longevity. In animals with reduced DAF-2/InsR activity induced by daf-2/InsR RNAi, both liganded and unliganded DAF-12 promote longevity. However, in daf-2/InsR mutants, liganded and unliganded DAF-12 act in opposition to control life span. Thus, multiple DAF-12 activities influence life span in distinct ways in contexts of reduced DAF-2/InsR signaling. Our findings establish new roles for a conserved steroid signaling pathway in life span control and elucidate interactions among DA biosynthetic pathways, DAF-12, and DAF-2/InsR signaling in aging.

Spermatogenic cell differentiation involves changes in the concentration of cytoplasmic Ca(2+) ([Ca(2+)]i); however, very few studies exist on [Ca(2+)]i dynamics in these cells. Other tissues display Ca(2+) oscillations involving multicellular functional arrangements. These phenomena have been studied in acute slice preparations that preserve tissue architecture and intercellular communications. Here we report the implementation of intracellular Ca(2+) imaging in a sliced seminiferous tubule (SST) preparation to visualize [Ca(2+)]i changes of living germ cells in situ within the SST preparation. Ca(2+) imaging revealed that a subpopulation of male germ cells display spontaneous [Ca(2+)]i fluctuations resulting from Ca(2+) entry possibly throughout Ca(V)3 channels. These [Ca(2+)]i fluctuation patterns are also present in single acutely dissociated germ cells, but they differ from those recorded from germ cells in the SST preparation. Often, spontaneous Ca(2+) fluctuations of spermatogenic cells in the SST occur synchronously, so that clusters of cells can display Ca(2+) oscillations for at least 10 min. Synchronous Ca(2+) oscillations could be mediated by intercellular communication via gap junctions, although intercellular bridges could also be involved. We also observed an increase in [Ca(2+)]i after testosterone application, suggesting the presence of functional Sertoli cells in the SST. In summary, we believe that the SST preparation is suitable to explore the physiology of spermatogenic cells in their natural environment, within the seminiferous tubules, in particular Ca(2+) signaling phenomena, functional cell-cell communication, and multicellular functional arrangements.

Glucose is an important monosaccharide required to generate energy in all cells. After entry into cells, glucose is phosphorylated to glucose-6-phosphate and then transformed into glycogen or metabolized to produce energy. Glucose phosphate isomerase (GPI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. Without GPI activity or fructose-6-phosphate, many steps of glucose metabolism would not occur. The requirement for GPI activity for normal functioning of primordial germ cells (PGCs) needs to be identified. In this study, we first examined the expression of chicken GPI during early embryonic development and germ cell development. GPI expression was strongly and ubiquitously detected in chicken early embryos and embryonic tissues at Embryonic Day 6.5 (E6.5). Continuous GPI expression was detected in PGCs and germ cells of both sexes during gonadal development. Specifically, GPI expression was stronger in male germ cells than in female germ cells during embryonic development and the majority of post-hatching development. Then, we used siRNA-1499 to knock down GPI expression in PGCs. siRNA-1499 caused an 85% knockdown in GPI, and PGC proliferation was also affected 48 h after transfection. We further examined the knockdown effects on 28 genes related to the glycolysis/gluconeogenesis pathway and the endogenous glucose level in chicken PGCs. Among genes related to glycolysis/gluconeogenesis, 20 genes showed approximately 3-fold lower expression, 4 showed approximately 10-fold lower, and 2 showed approximately 100-fold lower expression in knockdown PGCs. The endogenous glucose level was significantly reduced in knockdown PGCs. We conclude that the GPI gene is crucial for maintaining glycolysis and supplying energy to developing PGCs.

To assess the potential risks of human exposure to endocrine active compounds (EACs), the mechanisms of toxicity must first be identified and characterized. Currently, there are no robust in vitro models for identifying the mechanisms of toxicity in germ cells resulting from EAC exposure. Human embryonic stem cells can differentiate into numerous functional cell types including germ-like cells (GLCs). These cells possess characteristics indicative of a germ cell state, suggesting they offer a novel system to investigate the consequences of chemical exposure on normal germ cell processes. To characterize these processes, a metabolomic-based approach was employed to determine the response of GLCs following exposure to 0.001, 0.01, 0.1, 1, 10, or 100µM estradiol, testosterone, or progesterone for 48h. Following exposure, cellular extracts underwent gas chromatography coupled with mass spectrometry analysis. Models were then constructed using principal component analysis on acquired spectra to discriminate among steroid hormones as well as doses for each hormone. t-test comparisons generated a preliminary list of metabolites that were statistically significant in GLC's biochemical response to these steroid hormones. Steroid hormone exposures caused fluxes in intracellular pathways such as amino acid synthesis and metabolism, fatty acid synthesis, as well as cholesterol and lipoprotein metabolism. Further pathway analysis, based on these identified metabolites, will aid in modeling the response of GLCs to endogenous steroid hormones and allow for identification of biomarkers delineating germ cell-based developmental and reproductive pathways.

Historically, effects of environmental toxicants on human development have been deduced via epidemiological studies because direct experimental analysis has not been possible. However, in recent years, the derivation of human pluripotent stem cells has provided a potential experimental system to directly probe human development. Here, we used human embryonic stem cells (hESCs) to study the effect of environmental toxicants on human germ cell development, with a focus on differentiation of the founding population of primordial germ cells (PGCs), which will go on to form the oocytes of the adult. We demonstrate that human PGC numbers are specifically reduced by exposure to polycyclic aromatic hydrocarbons (PAHs), a group of toxicants common in air pollutants released from gasoline combustion or tobacco smoke. Further, we demonstrate that the adverse effects of PAH exposure are mediated through the aromatic hydrocarbon receptor (AHR) and BAX pathway. This study demonstrates the utility of hESCs as a model system for direct examination of the molecular and genetic pathways of environmental toxicants on human germ cell development.

Pituitary gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) act via their cognate glycoprotein hormone receptors (GpHRs), FSH receptor (FSHR), and LH/choriogonadotropin receptor (LHCGR) to regulate gonad physiology. Here, we show that the flatfish Senegalese sole (Solea senegalensis) expresses functional isoforms of fshr and lhcgr, but the genomic origin, ligand activation, and tissue distribution of the receptor transcripts are more complex than expected. By integrating the molecular phylogeny of GpHRs with the syntenic loci of vertebrate orthologs, and by subsequently characterizing the physical maps with the phylogeny of flanking genes, we found that vertebrate GpHRs have undergone a divergent evolution. In Teleostei, fshr genes have a common descent and can be classified as fshra, whereas lhcgrb genes exist as alternatively coded genes even in closely related species. Structural analyses of the receptors revealed that Fshra has an elongated ligand-binding domain, containing an extra leucine-rich repeat that specifically arose in the Acanthomorpha because of exon duplication. Ectopic expression in Xenopus laevis oocytes demonstrated that sole Fshra responded to piscine Fsh and Lh, whereas Lhcgrba was preferentially activated by its cognate hormone. The expression pattern of sole fshra and lhcgrba in gonads during the reproductive cycle was consistent with earlier observations wherein Fshra regulates ovarian growth and spermatogenesis and Lhcgrb triggers gamete maturation, respectively. However, contrary to observations in other teleosts, fshra was localized exclusively in Sertoli cells of the testis, whereas lhcgrba was expressed in Leydig cells as well as in spermatids. These results demonstrate the presence of alternatively coded lhcgr isoforms (lhcgrba and lhcgrbb) in teleosts and suggest a role of the lhcgrba receptor in the differentiation of spermatids into spermatozoa in Senegalese sole.

Modulating germ cell death and survival have significant therapeutic potential for male infertility and contraception. We have shown previously that IGF binding protein 3 (IGFBP3) gene expression is up-regulated in human testis when germ cell apoptosis is induced by intratesticular hormonal deprivation created by testosterone administration. Humanin (HN) is a binding partner of IGFBP3, and both are expressed in rat testes. We therefore hypothesized that IGFBP3, a proapoptotic factor, and HN, an antiapoptotic factor, are important regulators of male germ cell apoptosis. Whereas baseline apoptosis in the testis was equivalent between Igfbp3 knockout and wild-type mice, treatment with GnRH antagonist (GnRH-A) for 2 wk induced germ cell apoptosis in wild type, which was dramatically reduced in Igfbp3 knockout mice. To investigate the direct effects of IGFBP3 and HN on germ cell apoptosis, intratesticular administration of IGFBP3 for 5 d in rats induced a 4.2- and 3.8-fold increase in apoptosis at stages VII-VIII and XIV-I of the seminiferous epithelium cycle, respectively. GnRH-A treatment for 5 d increased apoptosis, mainly at stages VII-VIII. Addition of IGFBP3 to GnRH-A treatment enhanced apoptosis to 39.3-fold at stages VII-VIII, which was higher than either treatment alone. Intratesticular injection of HN significantly decreased GnRH-A-induced apoptosis at stages XIV-I but not stages VII-VIII. We conclude that IGFBP3 and HN play key roles in the coordinated regulation of testicular germ cell homeostasis. Perturbation of this interaction is important in enhancing or preventing germ cell death, providing new targets for future therapies.

Groups of male B6C3F1 mice were exposed by inhalation to 0, 25, 50, 100 or 200 p.p.m. ethylene oxide (EO) for up to 48 weeks (6 hours/day, 5 days/week). Animals were sacrificed at 6, 12, 24 and 48 weeks after the start of the exposure for analyses of reciprocal translocations in peripheral blood lymphocytes and germ cells. The frequency of the total chromosomal aberrations in the peripheral blood lymphocytes was significantly increased at the 100 and 200 p.p.m. exposure concentrations at the 12-week time point, at 50, 100 and 200 p.p.m. at the 24-week time point and at all EO concentrations at the 48-week time point. The frequency of stable reciprocal translocations, which can be used as biomarkers, was increased (P < 0.05) at 100 and 200 p.p.m. at the 12-week time point, at 100 and 200 p.p.m. at the 24-week time point and at 50, 100 and 200 p.p.m. at the 48-week time point. No statistically significant increase could be observed in translocation frequencies at the 6-week time point in the peripheral blood lymphocytes. The exposure-response curves were non-linear when the frequencies of translocations were plotted against EO exposure durations or against EO exposure concentrations. There was no effect of exposure concentration rate on reciprocal translocation frequency. Reciprocal translocations induced in spermatogonial stem cells (observed at the sprematocyte stage) showed significant increases in translocation frequencies over controls at all EO concentrations at 48 weeks. However, increases were small and they did not occur in a dose-responsive manner. The statistically significant increase observed at 12 weeks in the spermatocytes was equivocal. This study provides low-level chronic exposure somatic cytogenetic data generated in mice that can be used to support the shape of the tumour dose-response in rodents and humans The germ cell cytogenetic data are discussed in terms of its relevance for a threshold response for genetic effects at low exposures.

Although gonadotropins and androgen are required for normal spermatogenesis and both testosterone and follicle-stimulating hormone (FSH) are responsible for the inhibition of spermatogonial differentiation that occurs in irradiated rats, it has been difficult to identify the specific genes involved. To study specific hormonally regulated changes in somatic cell gene expression in the testis that may be involved in these processes, without the complication of changing populations of germ cells, we used irradiated LBNF(1) rats, the testes of which contain almost exclusively somatic cells except for a few type A spermatogonia. Three different groups of these rats were treated with various combinations of gonadotropin-releasing hormone antagonist, an androgen receptor antagonist (flutamide), testosterone, and FSH, and we compared the gene expression levels 2 wk later to those of irradiated-only rats by microarray analysis. By dividing the gene expression patterns into three major patterns and 11 subpatterns, we successfully distinguished, in a single study, the genes that were specifically regulated by testosterone, by luteinizing hormone (LH), and by FSH from the large number of genes that were not hormonally regulated in the testis. We found that hormones produced more dramatic upregulation than downregulation of gene expression: Testosterone had the strongest upregulatory effect, LH had a modest but appreciable upregulatory effect, and FSH had a minor upregulatory effect. We also separately identified the somatic cell genes that were chronically upregulated by irradiation. Thus, the present study identified gene expression changes that may be responsible for hormonal action on somatic cells to support normal spermatogenesis and the hormone-mediated block in spermatogonial development after irradiation.

Background: Fetal exposure of male rats to some phthalates induces reproductive abnormalities, raising concerns for similar effects in humans. In order to address this in a more appropriate animal model, the aim of the present studies was to investigate the effect of fetal/neonatal exposure to monobutyl phthalate (MBP) in a non-human primate, the marmoset. In particular, to determine if exposure resulted in effects at birth, or in adulthood, similar to those in male rats, and whether there was evidence for induction of carcinoma-in-situ (CIS) or testicular germ cell tumours (TGCT)., Methods: Pregnant female marmosets were dosed from approximately 7-15 weeks gestation with 500 mg/kg/day MBP and male offspring studied at birth (1-5 days; n = 6) or in adulthood (n = 5). In another study, newborn males (n = 5 co-twins) were dosed with 500 mg/kg/day MBP for 14 days, commencing at approximately 4 days of age., Results: Fetal exposure of marmosets to MBP did not affect gross testicular morphology, reproductive tract development or testosterone levels at birth, nor were germ cell number and proliferation, Sertoli cell number or germ:Sertoli cell ratio affected. In two of six MBP-exposed animals, unusual clusters of undifferentiated germ cells were found, but their significance is unclear. Neonatal MBP treatment did not affect germ cell numbers or differentiation. Fetal exposure to MBP did not affect testis size/morphology, germ cell numbers or fertility in adulthood. There was no evidence for CIS or TGCT., Conclusions: Fetal exposure of marmosets to MBP does not measurably affect testis development/function or cause testicular dysgenesis, and no effects emerge by adulthood. Some effects on germ cell development were found, but these were inconsistent and of uncertain significance.

Since the publication of the International Programme on Chemical Safety (IPCS) Harmonized Scheme for Mutagenicity Testing, there have been a number of publications addressing test strategies for mutagenicity. Safety assessments of substances with regard to genotoxicity are generally based on a combination of tests to assess effects on three major end points of genetic damage associated with human disease: gene mutation, clastogenicity and aneuploidy. It is now clear from the results of international collaborative studies and the large databases that are currently available for the assays evaluated that no single assay can detect all genotoxic substances. The World Health Organization therefore decided to update the IPCS Harmonized Scheme for Mutagenicity Testing as part of the IPCS project on the Harmonization of Approaches to the Assessment of Risk from Exposure to Chemicals. The approach presented in this paper focuses on the identification of mutagens and genotoxic carcinogens. Selection of appropriate in vitro and in vivo tests as well as a strategy for germ cell testing are described.

Lindane (gamma-HCH) was examined for its effect on primordial germ cell (PGC) development in the mouse embryo. We found that exposure by gavage of pregnant mice to 15 or 30 mg/kg/bw lindane during the period of PGC migration and gonad colonization (from 8.5 to 11.5 days post coitum, dpc) resulted in a significant reduction of the number of germ cells within 12.5 dpc testis and ovaries (a maximum of about 25 and 40%, respectively). Similarly, lindane caused a dose-dependent decrease of the PGC number in an in vitro culture model. Further experiments showed that in such model, lindane induced features of apoptotic cell death in PGCs such as increase in caspase-3 activity, poly-ADP-ribose polymerase cleavage, and terminal dUTP nick-end labeling (TUNEL) positivity. A marked increase of the number of PGCs positive for TUNEL staining was also observed in 12.5 dpc gonads of embryos from pregnant mice subjected one day before to acute lindane treatment (60 mg/kg/bw). Finally, we show that a brief incubation of isolated PGCs with 10(-5)M lindane resulted in a marked decrease of the basal and kit-ligand-induced phosphorylation level of the AKT kinase, known to be crucial for PGC survival. Taken together these results demonstrate that embryo exposure to lindane during early stages of gametogenesis can severely impair the number of germ cells in the fetal gonads; the compound appears to affect PGC survival through a direct proapoptotic action likely resulting from its adverse effect on AKT activity in such cells.

Epidermal growth factor (EGF) has been shown to stimulate survival in diverse cells in vitro. In the present study, the effects of EGF and the EGF-related signaling pathway on proliferation of chicken primordial germ cells (PGCs) were investigated. Results showed that EGF (10-100 ng/ml) increased the number and area of PGC colonies in a time- and dose-dependent manner. EGF also activated PKC, a process that was inhibited by AG1478 (an EGFR tyrosine kinase inhibitor) and ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA; an intracellular Ca(2+) chelator). In addition, the degradation of NFKBIA and NFKB1 (p65) translocation was observed after EGF treatment, which was significantly blocked by pretreatment with AG1478, EGTA, H(7), or SN50 (NFKB1-specific inhibitor). Furthermore, we found that EGF-induced cell proliferation was significantly attenuated by AG1478, EGTA, H(7), and SN50, respectively. On the other hand, inhibition of EGFR, Ca(2+)/PKC, or NFKB1 abolished the EGF-stimulated increase in the expression of cyclins CCND1 and CCNE1, cyclin-dependent kinase 6 (CDK6), CDK2, and BCL2, and restored the EGF-induced inhibition of BAX expression and caspase 3/9 activity, indicating that EGFR, PKC, and NFKB1 signaling cascades were involved in EGF-stimulated DNA synthesis and antiapoptosis action. In conclusion, EGF stimulated proliferation of chicken PGCs via activation of Ca(2+)/PKC involving NFKB1 signaling pathway. These observations suggest that EGF signaling is important in regulating germ cell proliferation in the chicken embryonic gonad.

Advances in treatment for testicular cancer that include the coadministration of bleomycin, etoposide, and cisplatin (BEP) have brought the cure rate to higher than 90%%. The goal of this study was to elucidate the impact of BEP treatment on gene expression in male germ cells. Brown-Norway rats were treated for 9 wk with vehicle (0x) or BEP at doses equivalent to 0.3x and 0.6x the human dose. At the end of treatment, spermatogenesis was affected, showing altered histology and a decreased sperm count; spermatozoa had a higher number of DNA breaks. After 9 wk of treatment, round spermatids were isolated, and RNA was extracted and probed on Rat230-2.0 Affymetrix arrays. Of the 31 099 probe sets present on the array, 59%% were expressed in control round spermatids. BEP treatment significantly altered the expression of 221 probe sets, with at least a 1.5-fold change compared with controls; 80% were upregulated. We observed a dose-dependent increase in the expression of oxidative stress response genes and no change in the expression of genes involved in DNA repair. BEP upregulated genes were implicated in pathways related to Jun and Junb protooncogenes. Increased mRNA levels of Jun and Junb were confirmed by quantitative RT-PCR; furthermore, JUN protein was increased in elongating spermatids. Thus, BEP exposure triggers an oxidative stress response in round spermatids and induces many pathways that may lead to the survival of damaged cells and production of abnormal sperm.

Notch signaling directs cell fate during embryogenesis by influencing cell proliferation, differentiation, and apoptosis. Notch genes are expressed in the adult mouse ovary, and roles for Notch in regulating folliculogenesis are beginning to emerge from mouse genetic models. We investigated how Notch signaling might influence the formation of primordial follicles. Follicle assembly takes place when germ cell syncytia within the ovary break down and germ cells are encapsulated by pregranulosa cells. In the mouse, this occurs during the first 4-5 d of postnatal life. The expression of Notch family genes in the neonatal mouse ovary was determined through RT-PCR measurements. Jagged1, Notch2, and Hes1 transcripts were the most abundantly expressed ligand, receptor, and target gene, respectively. Jagged1 and Hey2 mRNAs were up-regulated over the period of follicle formation. Localization studies demonstrated that JAGGED1 is expressed in germ cells prior to follicle assembly and in the oocytes of primordial follicles. Pregranulosa cells that surround germ cell nests express HES1. In addition, pregranulosa cells of primordial follicles expressed NOTCH2 and Hey2 mRNA. We used an ex vivo ovary culture system to assess the requirement for Notch signaling during early follicle development. Newborn ovaries cultured in the presence of gamma-secretase inhibitors, compounds that attenuate Notch signaling, had a marked reduction in primordial follicles compared with vehicle-treated ovaries, and there was a corresponding increase in germ cells that remained within nests. These data support a functional role for Notch signaling in regulating primordial follicle formation.

Human germ cell tumors are often metastatic, presumably due to distal site tumor growth by cancer stem cells. To determine whether cancer stem cells can be identified in a transplantation model of testicular germ cell tumor, we transplanted murine embryonic germ cells (EGCs) into the testis of adult severe combined immunodeficient mice. Transplantation resulted in a locally invasive solid tumor, with a cellular component that generated secondary tumors upon serial transplantation. The secondary tumors were invariably metastatic, a feature not observed in the primary tumors derived from EGCs. To characterize the differences between EGCs and the tumor-derived stem cells, we performed karyotype and microarray analysis. Our results show that generation of cancer stem cells is associated with the acquisition of nonclonal genomic rearrangements not found in the originating population. Furthermore, pretreatment of EGCs with a potent inhibitor of self-renewal, retinoic acid, prevented tumor formation and the emergence of these genetically unstable cancer stem cells. Microarray analysis revealed that EGCs and first- and second-generation cancer stem cells were highly similar; however, approximately 1,000 differentially expressed transcripts could be identified corresponding to alterations in oncogenes and genes associated with motility and development. Combined, the data suggest that the activation of oncogenic pathways in a cellular background of genetic instability, coupled with an inherent ability to self-renew, is involved in the acquisition of metastatic behavior in the cancer stem cell population of tumors derived from pluripotent cells.

(-)-10-epi-Axisonitrile-3, a spirocyclic sesquiterpene isocyanide obtained from the marine sponge Geodia exigua, immobilized sperm of sea urchin and starfish to block fertilization at the minimum effective concentration of 0.4 microg/ml. On the other hand, fertilized eggs developed normally to the gastrula stage in the presence of a 250-times higher concentration of the isocyanide. Analysis by (31)P NMR revealed an accumulation of phosphocreatine and a depletion of inorganic phosphate in the isocyanide-treated sperm, suggesting that (-)-10-epi-axisonitrile-3 inhibited the phosphocreatine shuttle participating in the high-energy phosphate metabolism, thereby immobilizing sperm to block fertilization. No analogs of (-)-10-epi-axisonitrile-3 containing different functionalities or isocyanides with different carbon skeleton exhibited such activity.

S(N)1-type alkylating agents such as N-ethyl-N-nitrosourea (ENU) are very potent mutagens. They act by transferring their alkyl group to DNA bases, which, upon mispairing during replication, can cause single base pair mutations in the next replication cycle. As DNA mismatch repair (MMR) proteins are involved in the recognition of alkylation damage, we hypothesized that ENU-induced mutation rates could be increased in a MMR-deficient background, which would be beneficial for mutagenesis approaches. We applied a standard ENU mutagenesis protocol to adult zebrafish deficient in the MMR gene msh6 and heterozygous controls to study the effect of MMR on ENU-induced DNA damage. Dose-dependent lethality was found to be similar for homozygous and heterozygous mutants, indicating that there is no difference in ENU resistance. Mutation discovery by high-throughput dideoxy resequencing of genomic targets in outcrossed progeny of the mutagenized fish did also not reveal any differences in germ line mutation frequency. These results may indicate that the maximum mutation load for zebrafish has been reached with the currently used, highly optimized ENU mutagenesis protocol. Alternatively, the MMR system in the zebrafish germ line may be saturated very rapidly, thereby having a limited effect on high-dose ENU mutagenesis.

The transition metal cadmium (Cd) has been shown to induce apoptosis in a variety of cell lines and tissues. Caspase activation of the tumor suppressor gene p53 and mitogen-activated protein kinase (MAPK) signaling cascades have been reported to be involved in Cd-induced apoptosis. However, the underlying pathways of Cd-induced apoptosis have not been clearly elucidated in the in vivo systems, primarily for the lack of appropriate animal models. The nematode Caenorhabditis elegans has been shown to be a good model to study basic biological processes, including apoptosis. In this study, we used the mutated alleles of C. elegans homologs of known mammalian genes that are involved in regulation of apoptosis. Sublethal doses of Cd exposure increased C. elegans germline apoptosis in a dose- and time-dependent manner. The loss-of-function mutations of DNA damage response (DDR) genes HUS1 and p53 exhibited significant increase in germline apoptosis under Cd exposure, and the depletion of p53 antagonist ABL1 significantly enhanced apoptosis. Cd-induced apoptosis was blocked in the loss-of-function alleles of both c-Jun N-terminal kinase (JNK) and p38 MAPK cascades, which behaved normally under gamma-irradiation. Our findings implicate that both JNK and p38 MAPK cascades participate in Cd-induced apoptosis. Together, the results of this study suggest the nonessential roles of the DDR genes hus1 and p53 in Cd-induced germline apoptosis and that the apoptosis occurs through the ASK1/2-MKK7-JNK and ASK1/2-MKK3/6-p38 signaling pathways in a caspase-dependent manner. Finally, our study demonstrates that C. elegans is a mammalian in vivo substitute model to study the mechanisms of Cd-induced apoptosis.

In Caenorhabditis elegans, exogenous dsRNA can elicit systemic RNAi, a process that requires the function of many genes. Considering that the activities of many of these genes are also required for normal development, it is surprising that exposure to high concentrations of dsRNA does not elicit adverse consequences to animals. Here, we report inducible phenotypes in attenuated C. elegans strains reared in environments that include nonspecific dsRNA and elevated temperature. Under these conditions, chromosome integrity is compromised in RNAi-defective strains harboring mutations in rsd-2 or rsd-6. Specifically, rsd-2 mutants display defects in transposon silencing, while meiotic chromosome disjunction is affected in rsd-6 mutants. RSD-2 proteins localize to multiple cellular compartments, including the nucleolus and cytoplasmic compartments that, in part, are congruent with calreticulin and HAF-6. We considered that the RNAi defects in rsd-2 mutants might have relevance to membrane-associated functions; however, endomembrane compartmentalization and endocytosis/exocytosis markers in rsd-2 and rsd-6 mutants appear normal. The mutants also possess environmentally sensitive defects in cell-autonomous RNAi elicited from transgene-delivered dsRNAs. Thus, the ultimate functions of rsd-2 and rsd-6 in systemic RNAi are remarkably complex and environmentally responsive.

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.

Male contraception has focused, to a great extent, on approaches that induce azoospermia or severe oligospermia through accelerated germ cell apoptosis. Understanding the specific steps in the germ cell apoptotic pathways that are affected by male contraceptives will allow more specific targeting in future contraceptive development. In this study, we have used a nonhuman primate model to characterize the key apoptotic pathway(s) in germ cell death after mild testicular hyperthermia, hormonal deprivation, or combined interventions. Groups of 8 adult (7- to 10-year-old) cynomolgus monkeys (Macaca fascicularis) received one of the following treatments: 1) two empty silastic implants; 2) two 5.5-cm testosterone (T) implants; 3) daily exposure of testes to heat (43 degrees C for 30 min) for 2 consecutive days; and 4) two T implants plus testicular heat exposure for two consecutive days. Testicular biopsies were performed before and at Days 3, 8, and 28 of treatment. Treatment with T, heat, or both led to sustained activation of both mitogen-activated protein kinase (MAPK) 1/3 and MAPK14. Activation of MAPK1/3 and MAPK14 were accompanied by an increase in B-cell leukemia/lymphoma (BCL) 2 levels in both cytosolic and mitochondrial fractions of testicular lysates (BAX levels remained unaffected) and cytochrome c and DIABLO release from mitochondria. These treatments also resulted in inactivation of BCL2 through phosphorylation at serine 70, thereby favoring the death pathway. We conclude that the serine phosphorylation of BCL2 and activation of the MAPK14-mediated mitochondria-dependent pathway are critical for male germ cell death in monkeys.

Embryonic exposure to the endocrine disruptor vinclozolin at the time of gonadal sex determination was previously found to promote transgenerational disease states. The actions of vinclozolin appear to be due to epigenetic alterations in the male germline that are transmitted to subsequent generations. Analysis of the transgenerational epigenetic effects on the male germline (i.e. sperm) identified 25 candidate DNA sequences with altered methylation patterns in the vinclozolin generation sperm. These sequences were identified and mapped to specific genes and noncoding DNA regions. Bisulfite sequencing was used to confirm the altered methylation pattern of 15 of the candidate DNA sequences. Alterations in the epigenetic pattern (i.e. methylation) of these genes/DNA sequences were found in the F2 and F3 generation germline. Therefore, the reprogramming of the male germline involves the induction of new imprinted-like genes/DNA sequences that acquire an apparent permanent DNA methylation pattern that is passed at least through the paternal allele. The expression pattern of several of the genes during embryonic development were found to be altered in the vinclozolin F1 and F2 generation testis. A number of the imprinted-like genes/DNA sequences identified are associated with epigenetic linked diseases. In summary, an endocrine disruptor exposure during embryonic gonadal sex determination was found to promote an alteration in the epigenetic (i.e. induction of imprinted-like genes/DNA sequences) programming of the male germline, and this is associated with the development of transgenerational disease states.

The germ cell lineage is a specified cell population that passes through a series of differentiation steps before giving rise, eventually, to either eggs or sperm. We have investigated the manner in which primordial germ cells (PGCs) are reprogrammed in vitro to form pluripotent stem cells in response to exogenous fibroblast growth factor-2 (FGF-2). The response is dependent on time of exposure and concentration of FGF-2. PGCs isolated in culture show a motile phenotype and lose any expression of a characteristic germ cell marker, mouse vasa homolog. Subsequently, some but not all of the cells show further changes of phenotype, accompanied by changes in expression of endogenous FGF-2 and up-regulation of its receptor, fibroblast growth factor receptor-3, in the nucleus. We propose that it is from this reprogrammed component of the now heterogeneous PGC population that pluripotent stem cells arise.

Background: Alpha6beta1 integrin has been proposed to act as a sperm receptor on the mouse oocyte by interacting with spermatozoon fertilin beta. We investigated, in humans, whether oocyte integrins could act similarly in gamete fusion, using a cyclic peptide containing the putative disintegrin-binding domain of human fertilin beta [cyclic FEE (cFEE)] and RGD peptide., Methods: Zona-free eggs were inseminated in the absence or presence of peptides. To maintain the membrane protein pattern, the zona pellucida was removed by microdissection. Immunofluorescence and confocal microscopy were used to detect integrin subunits on the oocyte., Results: Unexpectedly, cFEE alone increased human gamete fusion by 94% instead of inhibiting fertilization. Furthermore, cFEE together with RGD potentiated the RGD-induced inhibition of fertilization in a dose-dependent manner. The data suggested the hypothesis of integrin cross-talk, further supported by the co-localization of alpha6beta1 and alphavbeta3 integrins, the putative receptors of cFEE and RGD peptides, respectively., Conclusions: RGD-sensitive and -insensitive integrins may be associated in a multimolecular complex working as a sperm receptor on the human oocyte membrane. Supplementation of human IVF culture medium with cFEE peptide might improve fertilization rates in ART.

Context: Combination of a GnRH antagonist (acyline), types I and II, 5alpha-reductase inhibitor (dutasteride) or levonorgestrel (LNG) with testosterone (T) treatment may augment the suppression of spermatogenesis and intratesticular (iT) steroids., Objective: The objective of this study was to assess the effects of combined hormonal contraceptive regimens on germ cell populations and iT steroids., Design, Setting, and Participants: Twenty-nine normal health men enrolled in this prospective, randomized, 14-wk study at the University of Washington., Intervention(s): Twenty-two men (n = 5-6/group) received 8 wk of T enanthate (TE; 100 mg, i.m., weekly) combined with 1) 125 microg LNG daily, orally; 2) 125 microg LNG plus 0.5 mg dutasteride daily, orally; 3) 300 microg/kg acyline twice weekly, s.c.; or 4) 125 microg LNG daily, orally, plus 300 microg/kg acyline twice weekly, s.c. Subjects then underwent a vasectomy and testicular biopsy. Control men (n = 7) proceeded directly to surgery., Main Outcome Measure(s): The main outcome measures were germ cells and iT steroids [T, dihydrotestosterone, 3alpha- and beta-androstanediol (Adiol), and estradiol (E2)]., Results: High iT levels of all androgens (6- to 123-fold serum levels) and E2 (407-fold serum levels) were found in control men. iTT (1.9-2.6% control; P < 0.001) and iT3betaAdiol (16-34% control; P < 0.05) levels decreased with all treatments. iT dihydrotestosterone (13-29% control; P < 0.05) and iT3alphaAdiol (44-47% control; P < 0.05) levels decreased with all but the TE plus LNG treatment. iTE2 levels decreased only in the TE plus acyline group (28% control; P = 0.01). Germ cells from type B spermatogonia onward were suppressed, with no differences between groups found. Variable sites of impairment of germ cell progression were evident between men (spermagonial maturation, meiosis 1 entry, and spermiation). Other than a negative correlation between iT3alphaAdiol and haploid germ cell number (P < 0.006), no correlations between germ cell number and gonadotropins, sperm concentration, or iT steroids were found., Conclusions: A similar high testicular:serum gradient exists for E2 and T in normal men, and 8 wk of gonadotropin suppression markedly reduces iTT, with 5alpha-reduced androgens and E2 levels decreasing to a much lesser degree. The heterogeneity of the germ cell response, regardless of treatment, gonadotropins or iT steroids, points to the individual sensitivity of sites in germ cell development, which is worthy of additional exploration.

Metabolism of folate is essential for proper cellular function. Within the folate pathway, methylenetetrahydrofolate reductase (MTHFR) reduces 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a methyl donor for remethylation of homocysteine to methionine, the precursor of S-adenosylmethionine. S-adenosylmethionine is the methyl donor for numerous cellular reactions. In adult male mice, MTHFR levels are highest in the testis; this finding, in conjunction with recent clinical evidence, suggest an important role for MTHFR in spermatogenesis. Indeed, we show here that severe MTHFR deficiency in male mice results in abnormal spermatogenesis and infertility. Maternal oral administration of betaine, an alternative methyl donor, throughout pregnancy and nursing, resulted in improved testicular histology in Mthfr-/- offspring at Postnatal Day 6, but not at 8 mo of age. However, when betaine supplementation was maintained postweaning, testicular histology improved, and sperm numbers and fertility increased significantly. We postulate that the adverse effects of MTHFR deficiency on spermatogenesis, may, in part, be mediated by alterations in the transmethylation pathway and suggest that betaine supplementation may provide a means to bypass MTHFR deficiency and its adverse effects on spermatogenesis by maintaining normal methylation levels within male germ cells.

Phthalates have been shown to elicit contrasting effects on the testis and the liver, causing testicular degeneration and promoting abnormal hepatocyte proliferation and carcinogenesis. In the present study, we compared the effects of phthalates on testicular and liver cells to better understand the mechanisms by which phthalates cause testicular degeneration. In vivo treatment of rats with di-(2-ethylhexyl) phthalate (DEHP) caused a threefold increase of germ cell apoptosis in the testis, whereas apoptosis was not changed significantly in livers from the same animals. Western blot analyses revealed that peroxisome proliferator-activated receptor (PPAR) alpha is equally abundant in the liver and the testis, whereas PPAR gamma and retinoic acid receptor (RAR) alpha are expressed more in the testis. To determine whether the principal metabolite of DEHP, mono-(2-ethylhexyl) phthalate (MEHP), or a strong peroxisome proliferator, 4-chloro-6(2,3-xylindino)-2-pyrimidinylthioacetic acid (Wy-14,643), have a differential effect in Sertoli and liver cells by altering the function of RAR alpha and PPARs, their nuclear trafficking patterns were compared in Sertoli and liver cells after treatment. Both MEHP and Wy-14,643 increased the nuclear localization of PPAR alpha and PPAR gamma in Sertoli cells, but they decreased the nuclear localization of RAR alpha, as previously shown. Both PPAR alpha and PPAR gamma were in the nucleus and cytoplasm of liver cells, but RAR alpha was predominant in the cytoplasm, regardless of the treatment. At the molecular level, MEHP and Wy-14,643 reduced the amount of phosphorylated mitogen-activated protein kinase (activated MAPK) in Sertoli cells. In comparison, both MEHP and Wy-14,643 increased phosphorylated MAPK in liver cells. These results suggest that phthalates may cause contrasting effects on the testis and the liver by differential activation of the MAPK pathway, RAR alpha, PPAR alpha, and PPAR gamma in these organs.

Recent studies have demonstrated that GnRH-analogues can stimulate regeneration of spermatogenesis of rats when administered after testicular damages. Although the mechanism of this phenomenon has not been elucidated yet, stem cell factor (SCF) produced by Sertoli cells was proposed to mediate the effects of GnRH-analogues on spermatogonial proliferation and/or survival. In the present study, we quantitatively evaluated the proliferation of spermatogonia and addressed whether SCF mediates the effect of GnRH-analogue on spermatogonial proliferation, using a novel approach combining spermatogonial transplantation and laser confocal microscopic observation. In the first experiment, using wild-type mice as recipients for spermatogonial transplantation, the number of donor spermatogonia per 100 Sertoli cells in each spermatogenic colony was significantly higher in the experimental group of mice treated with leuprorelin, a GnRH-agonist, than that of the control group at 4 and 5 wk after transplantation. In the second experiment, Steel/Steeldickie (Sl/Sld) mutant mice, which lack expression of membrane bound form SCF, were used as recipients. As seen in the first experiment, the number of undifferentiated spermatogonia was significantly higher in leuprorelin-treated than in the control group. Since undifferentiated spermatogonia do not express the receptor of SCF, the present study clearly demonstrates that neither membrane-bound nor secreted forms of SCF are involved in the mechanism of GnRH-analogue's effect on spermatogonial proliferation and/or survival.

During spermatogenesis, cell-cell actin-based adherens junctions (AJs), such as ectoplasmic specializations (ESs), between Sertoli and germ cells undergo extensive restructuring in the seminiferous epithelium to facilitate germ cell movement across the epithelium. Although the mechanism(s) that regulates AJ dynamics in the testis is virtually unknown, Rho GTPases have been implicated in the regulation of these events in other epithelia. Studies have shown that the in vitro assembly of the Sertoli-germ cell AJs but not of the Sertoli cell tight junctions (TJs) is associated with a transient but significant induction of RhoB. Immunohistochemistry has shown that the localization of RhoB in the seminiferous epithelium is stage specific, being lowest in stages VII-VIII prior to spermiation, and displays cell-specific association during the epithelial cycle. Throughout the cycle, RhoB was localized near the site of basal and apical ESs but was restricted to the periphery of the nuclei in elongating (but not elongated) spermatids, spermatocytes, and Sertoli cells. However, RhoB was not detected near the site of apical ESs at stages VII-VIII. Furthermore, disruption of AJs in Sertoli-germ cell cocultures either by hypotonic treatment or by treatment with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) also induced RhoB expression. When adult rats were treated with AF-2364 to perturb Sertoli-germ cell AJs in vivo, a approximately 4-fold induction in RhoB in the testis, but not in kidney and brain, was detected within 1 h, at least approximately 1-4 days before germ cell loss from the epithelium could be detected by histological analysis. The signaling pathway(s) by which AF-2364 perturbed the Sertoli-germ cell AJs apparently began with an initial activation of integrin, which in turn activated RhoB, ROCK1, (Rho-associated protein kinase 1, also called ROKbeta), LIMK1 (LIM kinase 1, also called lin-11 isl-1 mec3 kinase 1), and cofilin but not p140mDia and profilin via phosphorylation. Immunoprecipitation and immunoblots revealed that the induction of LIMK1 was mediated via an increase in its phospho-Ser but not phospho-Tyr content. Furthermore, Y-27632 ([(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide, 2HCl]), a specific ROCK inhibitor, could effectively delay the AF-2364-induced germ cell loss from the seminiferous epithelium in vivo, illustrating that the integrin/RhoB/ROCK/LIMK pathway indeed plays a crucial role in the regulation of Sertoli-germ cell AJ dynamics. The fact that the RhoB pathway in the kidney and brain was not activated suggests that AF-2364 exerts its effects primarily at the testis-specific ES multiprotein complex structures between Sertoli cells and spermatids. In summary, this report illustrates that Sertoli germ cell AJ dynamics are regulated, at least in part, via the integrin/ROCK/LIMK/cofilin signaling pathway.