Effect of macrophage polarization on parasitic protection against type 1 diabetes mellitus.

Type 1 diabetes mellitus is a chronic disease caused by the destruction of pancreatic beta cells. Based on the hygiene hypothesis, a growing body of evidence suggests a negative association between parasitic infections and diabetes in humans and animal models. The mechanism of parasite-mediated prevention of type 1 diabetes mellitus may be related to the adaptive and innate immune systems. Macrophage polarization is a new paradigm for the treatment of type 1 diabetes mellitus, and different host macrophage subsets play various roles during parasite infection. Proinflammatory cytokines are released by M1 macrophages, which are important in the development of type 1 diabetes mellitus. Parasite-activated M2 macrophages prevent the development of type 1 diabetes mellitus and can influence the development of adaptive immune responses through several mechanisms, including Th2 cells and regulatory T cells. Here, we review the role and mechanism of macrophage polarization in parasitic protection against type 1 diabetes mellitus.

The effect of regulatory T cells in Schistosoma-mediated protection against type 2 diabetes.

In western societies, the prevalence of type 2 diabetes (T2D) is related to the hygiene hypothesis, which implies that reduced exposure to infectious factors results in a loss of the immune stimulation necessary to form the immune system during development. In fact, it has been reported that parasites, such as Schistosoma, can improve or prevent the development of T2D, which may be related to the activity of immune cells, including regulatory T cells (Tregs). Hence, Schistosoma, Tregs, and T2D share a close relationship. Schistosoma infection and the molecules released can lead to an increase in Tregs, which play an important role in the suppression of T2D. In this review, we provide an overview of the role of Tregs in the response to Schistosoma infection and the protective mechanism of Schistosoma-related molecular products against T2D.

The effects of helminth infections against type 2 diabetes.

Type 2 diabetes mellitus (T2D) is a prevalent inflammation-related disease characterized by insulin resistance and elevated blood glucose levels. The high incidence rate of T2D in Western societies may be due to environmental conditions, including reduced worm exposure. In human and animal models, some helminths, such as Schistosoma, Nippostrongylus, Strongyloides, and Heligmosomoides, and their products reportedly ameliorate or prevent T2D progression. T2D induces adaptive immune pathways involved in the inhibition of type 1 immune responses, promotion of type 2 immune responses, and expansion of regulatory T cells and innate immune cells, such as macrophages, eosinophils, and group 2 innate lymphoid cells. Among immune cells expanded in T2DM, type 2 immune cells and macrophages are the most important and may have synergistic effects. The stimulation of host immunity by helminth infections also promotes interactions between the innate and adaptive immune systems. In this paper, we provide a comprehensive review of intestinal helminths' protective effects against T2D.

Role of adenylate kinase 1 in the integument development of Schistosoma japonicum schistosomula.

Schistosomula antigens play an important role in the growth and development of Schistosoma japonicum. We investigated the role of S. japonicum adenylate kinase 1 (SjAK1) in the growth and development of schistosomula. Quantitative real-time PCR showed that SjAK1 mRNA was expressed in all schistosomula stages, but increased gradually with the development of S. japonicum schistosomula. Using immunohistochemical techniques, the AK1 protein was found to be mainly distributed in the tegument and in some parenchymal tissues of the schistosomula. Double-stranded RNA-mediated knockdown of AK1 reduced AK1 mRNA transcripts by more than 90%; western blot analysis demonstrated that AK1 protein expression decreased by 66%. Scanning electron microscopy following RNA-mediated AK1 knockdown demonstrated that the sensory papillae degenerated significantly. Transmission electron microscopy demonstrated that the mean thickness of the tegument in the SjAK1 interference group was lower than that in the negative control group. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) suggested that, compared with the negative control group, apoptosis increased in the interference group. These results show that AK1 may be involved in the growth and development of S. japonicum schistosomula, and thus may be a target when developing treatments for schistosomiasis.

Effects of programmed cell death protein 10 on fecundity in Schistosoma japonicum.

Programmed cell death protein 10 (PCDP10) is widely distributed in animal tissues and exerts extensive biological effects. This study aimed to investigate the effect of Schistosoma japonicum PCDP10 (SjPCDP10) on the fecundity of schistosomes. We performed real-time PCR to assess Sjpcdp10 expression levels at different developmental stages of S. japonicum. Immunoprotection against S. japonicum was assessed in vivo in mice, and Sjpcdp10 expression was inhibited via RNA interference (RNAi) to determine its role in fecundity. Real-time PCR analysis revealed that Sjpcdp10 mRNA was expressed during different developmental stages in S. japonicum, reaching maximum and minimum levels in female worms and lung-stage schistosomula, respectively. Recombinant SjPCDP10 had a molecular weight of approximately 28 kDa, displaying good immunogenicity but poor immunoprotection. SjPCDP10 was primarily localized in the egg, eggshell, epiphragm of adult worms, and especially the vitelline glands of female worms. RNAi-mediated knockdown of Sjpcdp10 by greater than 90%, and the protein expression decreased by 73%, reduced the number of eggs per female worm significantly more than RNAi-mediated knockdown of Egfp (negative control) (P < 0.05). The present results indicate that Sjpcdp10 knockdown affects the fecundity of schistosomes and may play a vital role in oogenesis.

Effects of programmed cell death protein 10 on the Schistosoma japonicum female reproductive system.

This study aimed to investigate the effects of programmed cell death protein 10 (PCDP10) on the female reproductive system of Schistosoma japonicum, one of the major infectious agents of schistosomiasis. We found that PCDP10 was widely distributed in the integument, the worm parenchymal area, and the vitellarium of the female worm, but was localized to a lesser extent in the ovary and testicles. RNAi experiments successfully achieved gene knockdown, and the ultrastructural morphology of the adult reproductive organs was observed. The results demonstrated that, compared with those of the negative control group, the number of cortical granules around oocytes decreased and the number of immature oocyte cells increased. Fusion of yolk globules occurred, and the number and the diameter of yolk droplets decreased significantly. Real-time PCR showed that the expression of yolk glands reached its peak before ovulation and then decreased. The TUNEL assay results showed that apoptosis in the RNAi group was significantly higher than that in the negative control group. These results suggested that SjPCDP10 plays an important role in the female reproductive system. In conclusion, PCD10 is involved in oocyte growth and development, especially in eggshell formation, which may provide a reference for further elucidating the molecular mechanism of PCDP10 involved in egg formation and embryo development in Schistosoma japonicum.

Role of regulatory T cells in Schistosoma-mediated protection against type 1 diabetes.

The prevalence of T1D in developed societies is partly based on the hygiene hypothesis, that is, the loss of exposure to infectious agents accompanies the loss of immune stimuli shaping the immune system during development. Indeed, the components of parasites, such as Schistosoma, have been reported to ameliorate or prevent the development of T1D, which might be associated with immune cell activity especially that of regulatory T cells (Tregs). Schistosoma infection can lead to the expansion of Treg. Herein, we provide a comprehensive overview of the involvement of Tregs in the response against Schistosoma infection and the mechanism of Schistosoma-associated host protection against T1D.

Influence of Schistosoma japonicum programmed cell death protein 10 on the growth and development of schistosomula.

BACKGROUND: Schistosomiasis caused by Schistosoma japonicum is among the most serious endemic zoonoses in China. To study interactions between schistosomula, the pre-adult juvenile stage, and hosts, it is important to study the functions of key genes involved in schistosomula growth and development. Programmed cell death protein 10 (pcdp10) is an important apoptosis-related gene with various biological functions. This study described the molecular characterization of S. japonicum PCDP10 (SjPCDP10) and evaluated its functions in schistosomula. METHODS: Real-time quantitative polymerase chain reaction (qPCR) and western blot were used to detect Sjpcdp10 mRNA and protein levels, respectively, at different developmental stages. Immunolocalization was performed to determine SjPCDP10 expression in the parasite. RNA interference (RNAi) experiments were used to assess gene functions associated with SjPCDP10 in schistosomula growth and development. RESULTS: Real-time qPCR revealed that Sjpcdp10 was expressed during all investigated developmental stages and upregulated during schistosomula growth and development. Histochemical localization showed that SjPCDP10 was mainly distributed in the teguments of schistosomula in all investigated stages and part of the parenchymal area of 14-, 18-, and 21-day-old schistosomula. Following Sjpcdp10 knockdown by RNAi, the lengths, widths, areas, and volumes of schistosomula were significantly lower than those in the control group. Scanning electron microscopy showed that the body surfaces of schistosomula subjected to RNAi were seriously damaged, with few tegumental spines and sensory papillae. Transmission electron microscopy indicated that the teguments of Sjpcdp10-knockdown schistosomula were incomplete, the number of layers was reduced, and the thickness decreased significantly as compared with those in the control group. Furthermore, terminal deoxynucleotidyl transferase dUTP nick-end labelling results showed that the rate of apoptosis in Sjpcdp10-knockdown schistosomula was significantly higher than that in the control group. CONCLUSIONS: Sjpcdp10-knockdown influenced the growth and development of schistosomula. Therefore, our results indicated that SjPCDP10 contributes to the regulation of cell apoptosis and is essential for schistosomula growth and development.

Schistosoma Infection and Schistosoma-Derived Products Modulate the Immune Responses Associated with Protection against Type 2 Diabetes.

Studies on parasite-induced immunoregulatory mechanisms could contribute to the development of new therapies for inflammatory diseases such as type 2 diabetes (T2D), which is a chronic inflammatory disease characterized by persistent elevated glucose levels due to insulin resistance. The association between previous Schistosoma infection and T2D has been confirmed-Schistosoma infection and Schistosoma-derived products modulate the immune system, including innate and acquired immune responses, contributing to T2D disease control. Schistosoma infections and Schistosoma-derived molecules affect the immune cell composition in adipose tissue, dampening inflammation and improving glucose tolerance. This protective role includes the polarization of immune cells to alternatively activated macrophages, dendritic cells, eosinophils, and group 2 innate lymphoid cells. Furthermore, Schistosoma infection and Schistosoma products are effective for the treatment of T2D, as they increase the number of type 2 helper T cells (Th2) and regulatory T cells (Tregs) and decrease type 1 helper T cells (Th1) and type 17 helper T cells (Th17) cells. Thus, our aim was to comprehensively review the mechanism through which Schistosoma infection and Schistosoma products modulate the immune response against T2D.

Androgen actions in mouse wound healing: Minimal in vivo effects of local antiandrogen delivery.

The aims of this work were to define the role of androgens in female wound healing and to develop and characterize a novel wound dressing with antiandrogens. Androgens retard wound healing in males, but their role in female wound healing has not been established. To understand androgen receptor (AR)-mediated androgen actions in male and female wound healing, we utilized the global AR knockout (ARKO) mouse model, with a mutated AR deleting the second zinc finger to disrupt DNA binding and transcriptional activation. AR inactivation enhanced wound healing rate in males by increasing re-epithelialization and collagen deposition even when wound contraction was eliminated. Cell proliferation and migration in ARKO male fibroblasts was significantly increased compared with wild-type (WT) fibroblasts. However, ARKO females showed a similar healing rate compared to WT females. To exploit local antiandrogen effects in wound healing, while minimizing off-target systemic effects, we developed a novel electrospun polycaprolactone (PCL) scaffold wound dressing material for sustained local antiandrogen delivery. Using the antiandrogen hydroxyl flutamide (HF) at 1, 5, and 10 mg/mL in PCL scaffolds, controlled HF delivery over 21 days significantly enhanced in vitro cell proliferation of human dermal fibroblasts and human keratinocytes. HF-PCL scaffolds also promoted in vivo wound healing in mice compared with open wounds but not to PCL scaffolds.

Androgen receptor actions modify skin structure and chemical carcinogen-induced skin cancer susceptibility in mice.

Men are significantly more susceptible to non-melanoma skin cancers than women, and the androgen receptor (AR) is widely distributed in the skin, suggesting a ro\le for androgens acting via AR. Therefore, we explored the role of androgen action via AR in susceptibility to experimental 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin carcinogenesis and in skin structural development of male and female mice. We demonstrate that both the male gender and androgen action via AR modify the susceptibility to carcinogen-induced skin cancer, but the effect depends on the carcinogenesis model used. Following systemic DMBA exposure, males were significantly (p < 0.05) more susceptible to DMBA-induced experimental skin cancer than females and AR inactivation significantly delayed cancer detection in both male (median time to palpable tumours 19 vs. >35 weeks (wild-type [WT] vs. AR knockout [ARKO], p < 0.001) and female (27 vs. >35 weeks, p = 0.008)) mice. In contrast, following DMBA/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced multistage local skin carcinogenesis, AR inactivation protected against formation of DMBA-induced skin cancers in both male and female mice. The skin structure was also affected by gender effect as well as the AR inactivation and could at least partly explain the different responses between the carcinogenesis models (systemic vs. topical). In addition, AR inactivation modified Cox-1 and Cox-2 expression in the skin, suggesting possible molecular mechanism for the AR effect on skin. Finally, some gender differences are observed also in ARKO mice insensitive to androgens, suggesting that factors other than androgens also play a role in gender-dependent skin carcinogenesis.

Androgen receptor inactivation resulted in acceleration in pubertal mammary gland growth, upregulation of ERα expression, and Wnt/ß-catenin signaling in female mice.

The androgen receptor (AR) is widely expressed in mammary cells of female mammals including humans and mice, indicating a possible role for AR-mediated androgen actions in breast development, function, and pathology, although the specific mechanisms remain unclear. To elucidate the mechanisms of androgen action in mammary gland physiology and development, we used AR-knockout (AR(Δex3)KO) female mice with a universally expressed, transcriptionally inactive AR protein harboring an in-frame deletion of its second zinc finger. Although in sexually mature wild-type (WT) and AR(ex3Δ)KO females, the mammary epithelial growth was fully extended to the edge of the fat pad, during puberty, AR(ex3Δ)KO females exhibit significantly accelerated mammary ductal growth and an increased number of terminal end buds compared with WT females. Accelerated AR(ex3Δ)KO female mammary growth was associated with significantly increased mammary epithelial ERα expression and activated Wnt/ß-catenin signaling as shown by increased Wnt4 expression and accumulation of nuclear ß-catenin. These findings are consistent with increased mammary estrogen exposure although ovarian estradiol content was unchanged compared with WT females. Furthermore, treatment with the potent pure androgen DHT markedly reduced ductal extension and terminal end bud numbers in WT but not in AR(Δex3)KO females, further supporting the concept that AR-mediated, androgen-induced suppression of murine mammary growth is a physiological characteristic of puberty. In summary, our findings reveal an inhibitory role of AR-mediated androgen actions in pubertal mammary gland development by reducing epithelial cell proliferation and could be mediated by regulation of Wnt/ß-catenin signaling.

Glucocorticoid receptor in prostate epithelia is not required for corticosteroid-induced epithelial hyperproliferation in the mouse prostate.

BACKGROUND: Glucocorticoids are used as a last resort treatment for prostate cancer but the cell-specific glucocorticoid receptor (GR) mediated actions and the role of endogenous glucocorticoids in prostate are not understood. METHODS: We evaluated the influence of prostate epithelial GR mediated actions of glucocorticoids in prostate structural development by comparing the intact wild-type (WT) and prostate epithelia selective GR knockout (peGRKO) males at 8, 20, and 35 weeks of age. We also determined the cell-specific role of GR on corticosterone treatment induced prostate abnormalities by treating peGRKO and WT male mice with corticosterone depot pellets or placebo for 4 weeks. RESULTS: GR was not expressed in the epithelial cells of peGRKO prostate unlike WT but was expressed in stromal of both peGRKO and WT mice. Nevertheless, prostate weights, histological appearance, and secretory protein probasin expression in peGRKO were no different from WT. Despite lacking epithelial GR, the peGRKO prostate demonstrated corticosterone treatment induced hyperplasia similar to WT suggesting that stromal rather than epithelial GR mediates the hyperproliferative mouse prostate response to corticosterone. As circulating androgen levels were not affected by corticosterone treatment, this effect is likely to be mediated directly via prostate GR. CONCLUSIONS: Sustained administration of corticosterone induces prostate hyperplasia, which is mediated via GR expressed predominantly in the stroma. Thus GR mediated actions in the prostate may have significant cell-specific effects that could be utilized for more rational therapeutic approaches in prostate cancer treatment. This also illustrates the paracrine hormonal mechanisms in prostate pathophysiology.

The role of androgens in experimental rodent mammary carcinogenesis.

Breast cancer is currently the most frequent, fatal cancer of women in western countries. While estrogens have a widely understood involvement in breast cancer, a significant but not yet fully understood role for androgens has also been suggested. The principal androgen, testosterone, is the obligate steroidal precursor of estradiol, but can equally be metabolized into dihydrotestosterone, a more potent, pure androgen. Both androgens exert their distinctive biological effects via the androgen receptor, which is coexpressed with estrogen receptor alpha in 80 to 90% of breast cancers. The hormonal control of breast development and pathology has been examined experimentally through the use of animal models, notably mice and rats. This review summarizes the data from experimental rodent models on the effects of androgens in experimental breast cancer, aiming to address the importance of androgens and the androgen receptor in the origins and pathogenesis of breast cancers, as well as to discuss potential biomarker and therapeutic opportunities arising from novel insights based on the experimental research.

Evidence for increased tissue androgen sensitivity in neurturin knockout mice.

Neurturin (NTN) is a member of the glial cell line-derived neurotrophic factor (GDNF) family and signals through GDNF family receptor alpha 2 (GFRα2). We hypothesised that epithelial atrophy reported in the reproductive organs of Ntn (Nrtn)- and Gfrα2 (Gfra2)-deficient mice could be due to NTN affecting the hormonal environment. To investigate this, we compared the reproductive organs of Ntn- and Gfrα2-deficient male mice in parallel with an analysis of their circulating reproductive hormone levels. There were no significant structural changes within the organs of the knockout mice; however, serum and intratesticular testosterone and serum LH levels were very low. To reconcile these observations, we tested androgen sensitivity by creating a dihydrotestosterone (DHT) clamp (castration plus DHT implant) to create fixed circulating levels of androgens, allowing the evaluation of androgen-sensitive endpoints. At the same serum DHT levels, serum LH levels were lower and prostate and seminal vesicle weights were higher in the Ntn knockout (NTNKO) mice than in the wild-type mice, suggesting an increased response to androgens in the accessory glands and hypothalamus and pituitary of the NTNKO mice. Testicular and pituitary responsiveness was unaffected in the NTNKO males, as determined by the response to the human chorionic gonadotrophin or GNRH analogue, leuprolide, respectively. In conclusion, our results suggest that NTN inactivation enhances androgen sensitivity in reproductive and neuroendocrine tissues, revealing a novel mechanism to influence reproductive function and the activity of other androgen-dependent tissues.

Androgen resistance in female mice increases susceptibility to DMBA-induced mammary tumors.

Hormones, notably estrogens, are pivotal in the origins of breast cancer but androgenic effects, while supported by persistence of AR expression in breast cancers, remain controversial. This study determined the role of the androgen actions via androgen receptor (AR) in experimental mammary cancer. Androgen-resistant female and male mice (ARKO) were generated using Cre/loxP technique and featured a global AR inactivation. The effect of AR inactivation and influence of genetic background on 7,12-dimethylbenz[a]anthracene (DMBA)-induced tumorigenesis was confirmed using two separate ARKO models with different genetic backgrounds. The onset of palpable mammary tumors was significantly faster in ARKO females (median time 22 vs 34 weeks, respectively; (p = 0.0024; multivariate Cox regression) compared to WT and independent of the mouse genetic background. The cumulative incidence at 9 months was 81 ± 10% [mean ± SE] for ARKO compared to 50 ± 13% in WT females. The increased DMBA susceptibility of ARKO females was associated with a higher epithelial proliferation index but not with major structural or receptor (estrogen or progesterone) expression differences between the virgin WT or ARKO female mammary glands. AR inactivation allowed substantial ductal extension in ARKO males while WT males displayed only rudimentary epithelial branches or complete regression of epithelial structures. Yet, DMBA did not induce epithelial mammary tumors in WT or ARKO males, demonstrating that AR inactivation alone is insufficient to promote mammary tumors. These results demonstrate that AR inactivation accelerates mammary carcinogenesis in female mice exposed to the chemical carcinogen DMBA regardless of mouse genetic background but require prior exposure to endogenous ovarian hormones.

Anterior prostate epithelial AR inactivation modifies estrogen receptor expression and increases estrogen sensitivity.

Androgens influence prostate growth and development, so androgen withdrawal can control progression of prostate diseases. Although estrogen treatment was originally used to induce androgen withdrawal, more recently direct estrogen effects on the prostate have been recognized, but the nature of androgen-estrogen interactions within the prostate remain poorly understood. To characterize androgen effects on estrogen sensitivity in the mouse prostate, we contrasted models of castration-induced androgen withdrawal in the prostate stromal and epithelial compartments with a prostate epithelial androgen receptor (AR) knockout (PEARKO) mouse model of selective epithelial AR inactivation. Castration markedly increased prostate epithelial estrogen receptor (ER)α immunoreactivity compared with very low ERα expression in intact males. Similarly, strong basal and luminal ERα expression was detected in PEARKO prostate of intact males, suggesting that epithelial AR activity regulated epithelial ERα expression. ERß was strongly expressed in intact, castrated, and PEARKO prostate. However, strong clusters of epithelial ERß positivity coincided with epithelial stratification in PEARKO prostate. In vivo estrogen sensitivity was increased in PEARKO males, with greater estradiol-induced prostate growth and epithelial proliferation leading to squamous metaplasia, featuring markedly increased epithelial proliferation, thickening, and keratinization compared with littermate controls. Our results suggest that ERα expression in the prostate epithelial cells is regulated by local, epithelia-specific, androgen-dependent mechanisms, and this imbalance in the AR- and ER-mediated signaling sensitizes the mature prostate to exogenous estrogens.

Length of the human androgen receptor glutamine tract determines androgen sensitivity in vivo.

A well established functional polymorphism of the human androgen receptor (hAR) is the length of AR's N-terminal glutamine tract (Q-tract). This tract is encoded by a CAG trinucleotide repeat and varies from 8 to 33 codons in the healthy population. Q-tract length is inversely correlated with AR transcriptional activity in vitro, but whether endogenous androgen action is affected is not consistently supported by results of clinical and epidemiological studies. To test whether Q-tract length influences androgen sensitivity in vivo, we examined effects of controlled androgen exposure in "humanized" mice with hAR knock-in alleles bearing 12, 21 or 48 CAGs. Mature male mice were analyzed before or 2weeks after orchidectomy, with or without a subdermal dihydrotestosterone (DHT) implant to attain stable levels of this non-aromatizable androgen. The validity of this DHT clamp was demonstrated by similar serum levels of DHT and its two primary 3αDiol and 3ßDiol metabolites, regardless of AR Q-tract length. Q-tract length was inversely related to DHT-induced suppression of castrate serum LH (p=0.005), as well as seminal vesicle (SV) weight (p=0.005) and prostate lobe weights (p<0.006). This confirms that the hAR Q-tract polymorphism mediates in vivo tissue androgen sensitivity by impacting negative hypothalamic feedback and trophic androgen effects on target organs. In this manner, AR Q-tract length variation may influence numerous aspects of male health, from virilization to fertility, as well as androgen-dependent diseases, such as prostate cancer.

Androgen sensitivity of prostate epithelium is enhanced by postnatal androgen receptor inactivation.

Postnatal inactivation of epithelial androgen receptor (AR) in prostate epithelial AR knockout (PEARKO) mice results in hindered differentiation but enhanced proliferation of epithelial cells. As this resembles the precancerous proliferative atrophy of human prostates with undifferentiated but intensively replicating epithelial cells, we utilized the PEARKO mice to characterize the epithelial response to castration-induced involution with a focus on identifying the potential role of stromal AR and responsiveness of the androgen-deprived epithelia to the aromatizable androgen testosterone (T) or its nonaromatizable metabolite dihydrotestosterone (DHT). PEARKO and littermate control mice were orchidectomized at 8 wk of age and treated 2 wk later with subdermal implantation of 1-cm Silastic tubing filled with T or DHT for a week. Following castration, the prostatic involution and epithelial apoptosis did not significantly differ between control (intact AR) and PEARKO (only stromal AR) males, demonstrating that prostate epithelial involution following castration is mediated primarily via stromal AR-dependent apoptotic signals. Androgen replacement (T/DHT) for 7 days induced significant growth and epithelial proliferation in all prostate lobes in both control and PEARKO, but full regrowth was observed only in controls treated with T. In PEARKO, prostate androgen (T and DHT) treatment induced significant epithelial cell "shedding" into the lumen, with T treatment resulting in acinar disorganization, cyst formation, and aberrant epithelial structures, described as a "gland within a gland." These data suggest that epithelial AR inactivation during postnatal prostate development sensitizes prostate epithelial cells to paracrine signaling mediated by stromal AR activity leading to indirectly androgen-induced epithelial hyperproliferation and formation of epithelial hyperplastic cysts by aromatizable androgens.

[Secretory expression of recombinant porcine zona pellucida glycoprotein-3alpha (rpZP3alpha) in Pichia pastoris].

To obtain the recombinant pZP3alpha protein for the study of the contraceptive vaccines, the DNA sequence (446-1423) encoding purified pZP3alpha was inserted into a vector--pPICZalphaA. The recombinant plasmid pPICZalphaA-pZP3alpha was linearized and then transformed into Pichia pastoris GS115 by electroporation. Engineering strains were attained by screening with zeocin and induced to produce rpZP3alpha in high-density fermentation. Then rpZP3alpha was purified by Cu2+ metal affinity column chromatography from the separated and concentrated fermentative supernatants. The purified rpZP3alpha was identified by SDS-PAGE and Western blot, and the quantity, purity and rate of recovery of the rpZP3alpha were analyzed by Quantity One software. One male rabbit was immunized with the Cu-NTA-purified rpZP3alpha. The antibody responses against rpZP3alpha and porcine ZP were detected by ELISA and the indirect immunofluorescence. Engineering strains expressing rpZP3alpha in secretion were constructed. A 46kD component named rpZP3alpha which can react with anti-pZP3 antibody was purified from fermentative supernatants of engineering strains and the average yield of purified rpZP3alpha obtained from fermentative supernatants was 8mg/L. The purity and the rate of recovery were up to 92% and 63% respectively. The anti-rpZP3alpha antiserum was prepared by immunization of a male rabbit with purified rpZP3alpha. This anti-rpZP3alpha antiserum could react with rpZP3alpha and purified pZP3 in ELISA and bind to porcine zona pellucida which produced bright green fluorescence in the indirect immunofluorescence. The rpZP3alpha (46kD) protein could be successfully expressed in the Pichia pastoris expression system. And this protein retained the immunogenic activity of natural pZP3.