Overexpression of aromatase leads to development of testicular leydig cell tumors : an in vivo model for hormone-mediated TesticularCancer

Despite recent advances in diagnosis and treatment of testicular cancer, its causes remain unknown. The most common conditions known to be associated with testicular cancer are cryptorchidism, infertility, and overexposure to pesticides or radiation. Recent studies also indicate hormones may play a crucial role in testicular tumorigenesis. Our studies show that about half of the male transgenic mice overexpressing aromatase in testis were infertile and/or had larger than normal testicles. Gross pathology and histological analysis showed the mice to have Leydig cell tumors, unilaterally or bilaterally. Serum estradiol levels for transgenic mice were at least twice as high as those for nontransgenic mice. Expression of aromatase and estrogen receptor were also very high in testicular tissue of transgenic mice compared to nontransgenic mice. Consistent with increased estrogenic activity in the testicular tissue, we also saw an increase in the levels of genes involved in cell cycle that are regulated by the estrogen. To obtain a better understanding of the biological significance of testicular tumorigenesis, a reliable animal model is necessary to clarify the mechanisms and correlations associated with human cancers. Here we describe such a model, which shows that overexpression of aromatase results in increased estrogen production and a changed hormone milieu, leading to the induction of testicular cancer (Leydig cell tumors). This predictable and useful model is a potential tool for the study of testicular tumorigenesis, hormonal carcinogenesis, synergistic action of other carcinogens on hormone-induced tumors, and tumor dependency on endocrine factors.     

On the origin of natural IgM in immunoglobulin transgenic mice.

Surface transgenic IgM was expressed by > 95% of small resting splenic B cells but only by 50% of CD5+ and CD5- peritoneal B cells from the mu-transgenic mouse line M54. Transgenic male M54 were crossed with female CBA/N mice carrying the Xid defect. Offspring F1 animals carrying the transgene were analysed for the presence of transgenic and endogenous IgM expressed both in the serum as well as on the surface of splenic and peritoneal B cells. We found that the levels of serum IgM coded for by the transgene were similar in both F1 male, which lack CD5 B cells, and female transgenic mice, which have CD5 B cells. Thus, the Xid defect does not influence the expression of the transgene at the level of naturally activated plasma cells, a finding substantiated by the fact that both male and female naturally activated splenic plasma cells express the transgene at the same frequency. F1 hybrid mice, like transgenic C57BI/6 M54 mice, have naturally activated splenic plasma cells that overexpress endogenous IgM coded for by the VH gene family Q52. The data indicate that normal serum IgM is not derived from CD5+ B cells and that the serum IgM coded for by the mu-transgene from M54 is produced at normal levels even in the male F1 mouse which lacks CD5+ B cells.     

Leydig cell tumor of the testis: comparison of histopathological and immunohistochemical features of three azoospermic cases and one malignant case

Leydig cell tumors of the testis are rare, mostly presenting as a testicular mass or as endocrinological symptoms. Here, three patients who were admitted for investigation of primary infertility and one patient presenting with a testicular mass are reported. The histological features were reviewed and an immunohistochemical study was done using a panel of antibodies against cytokeratin, vimentin, inhibin A, S-100, Ki-67, follicle-stimulating hormone, luteinizing hormone, prolactin, p53, bcl-2, and c-erbB2. The latter case (lost during follow up of metastatic disease) demonstrated massive tumor necrosis, extension through the tunica albuginea, and a high mitotic activity and MIB-1 score. Only this malignant case was bcl-2 positive. Of the two oncogenic markers studied, none of the cases were positive for c-erb2, while p53 was positive in more than 50% of cells in the malignant case and in one case of infertility with a large tumor, hemorrhage, focal necrosis and atypical cytological features. We recommend the evaluation of infertile men for Leydig cell tumors, and we believe that a panel of antibodies, including Ki-67, p53 and bcl-2, used for immunohistochemical analysis could be of diagnostic value in the identification of malignant and borderline cases of Leydig cell tumor.     

Reduced oocyte activation and first cleavage rate after ICSI with spermatozoa from a sterile mouse chromosome mutant

BACKGROUND: Male mice, heterozygous for two semi-identical reciprocal translocations T(1;13)70H and T(1;13)1Wa are usually sterile. We have investigated this oligoasthenoteratozoospermic mouse model using ICSI. METHODS: B6D2F1 oocytes were injected with epididymal or testicular sperm from fertile or sterile translocation carriers and from chromosomally normal fertile controls. ICSI efficiency was determined by pronucleus formation and first cleavage rates. For arrested zygotes, cell cycle progression was evaluated by BrdU incorporation and incubation with okadaic acid. RESULTS: Epididymal sperm from infertile translocation carriers showed a slightly lower fertilization rate (70% vs. 92%, 95% and 95% for fertile translocation carriers and two groups of normal fertile control males, respectively) and a severely reduced cleavage rate (33% vs. 87%, 96% and 89% for the same control groups). However, the use of testicular sperm significantly improved the cleavage rate (62% vs. 83% for normal fertile controls). Development of arrested zygotes was delayed or blocked during S- and G2-phase. CONCLUSIONS: Whereas control testicular and epididymal sperm performed equally well, the use of testicular sperm from oligospermic T/T' males significantly increased first cleavage rates when compared to the low rates with epididymal sperm. Epididymal storage in oligospermics may negatively influence zygote division.     

Infertility: Infertility evaluation in fertile women: a model for assessing the efficacy of infertility testing

A standard infertility evaluation consists of a semen analysis,hysterosalpingogram, post-coital test, endometrial biopsy andlaparoscopy. Although these tests are well grounded in clinicalexperience, information on their ability to discriminate betweenfertile and infertile couples is limited. In this study, weperformed standard infertility tests plus two others-sperm antibodiesand cervical culture for Mycoplasma hominis and Ureaplasma urealyticum–onfertile and infertile couples. Women in the fertile group wereselected from those who had delivered a child within the previous2 years and who were scheduled for a laparoscopic tubal ligation.Women in the infertile group were selected from those presentingfor an infertility evaluation (mean duration of infertility4.2 years), and they were matched by age (±3 years) andrace with fertile subjects. Subjects were recruited from bothprivate and clinic patients. A total of 64 couples (32 matchedpairs) completed the evaluation. At least one ’abnormal‘infertility test was found in 69% of fertile and 84% of infertilecouples. With the exception of tubal damage and endometriosis,which as expected were more common in infertile couples, nosignificant differences between groups for remaining infertilityfactors could be demonstrated. Despite the small size of thecurrent study, these results confirm the feasibility and importanceof comparisons of the prevalence of infertility factors in fertileand infertile couples.     

Lymphoid tissue in the endometrium of women with unexplained infertility: morphometric and immunohistochemical aspects

The purpose of this study was to investigate whether the endometriumof women with unexplained infertility differs in some immunologicalaspects from the endometrium of normal fertile women. Endometrialbiopsies were obtained from 24 normal fertile women (group I)and 24 women suffering from unexplained infertility (group II)at 4, 7, 10 and 13 days following the luteinizing hormone (LH)surge. Endometrial granulated lymphocytes were assessed morphometricallyin 2µm resin sections. A panel of 11 monoclonal antibodieswas employed to characterize the leukocyte subsets in frozensections. Semi-quantification was performed with a Quantimet970 image analyser. Data were analysed using one-and two-wayanalysis of variance. Compared with fertile controls, womenwith unexplained infertility had significantly lower numbersof CD8+ (T suppressor/cytotoxic) cells at each post-LH date.In contrast, the number of CD4+ (T helper/inducer) cells wassignificantly higher in group II. Throughout the luteal phase,infertile women had fewer CD56+ cells than normal fertile controls.The volume fraction of endometrium occupied by the nuclei ofendometrial granulated lymphocytes did not alter with the cyclestage but the mean nuclear diameter and axial ratio decreasedfrom LH+7 to LH+13. The differences observed in endometrialleukocytic subpopulations between fertile and infertile womenmay contribute to unexplained infertility probably by affectingthe embryonic maternal dialogue during the implantation andearly placentation period.     

Ubiquitous expression of human SCA2 gene under the regulation of the SCA2 self promoter cause specific Purkinje cell degeneration in transgenic mice

The objective of this work was the generation of an animal model of the SCA2 disease for future studies on the benefits of therapeutic molecules and neuropathological mechanisms that underline this human disorder. The transgenic fragment was microinjected into pronuclei of B6D2F1 X OF1 mouse hybrid strain. For Northern blots, RNAs were hybridized with a human cDNA fragment from the SCA2 gene and a mouse beta-actin cDNA fragment. Monoclonal antibody directed to the N-terminal of the ataxin 2 protein with 22Q was used for Western blot analysis. A rotating rod apparatus was utilized to measure motor coordination of mice. Immunohistochemical detection of Purkinje neurons was performed with anti-calbindin 28K as primary antibody. Ubiquitous expression of the SCA2 transgene with 75 CAG repeats regulated by the SCA2 self promoter was obtained after generation of our transgenic mice. Analysis of transgenic mice revealed significant differences of motor coordination compared with the wild type littermates. Specific degeneration of Purkinje neurons and transgene over-expression in the brain, liver and skeletal muscle, rather than in lungs and kidneys was also observed, resembling the expression pattern of the ataxin 2 in humans.     

Importance of the testicular artery: histo-functional approach and comparison between juvenile and adult rats

The aim of this study is to demonstrate the role of spermatic or testicular artery with regard to fertility. 100 male rats Sprague-Dawley, consisted of 50 young rats (aged from 10 to 12 days old) and 50 adult rats were concerned. Unilateral ligation of the testicular artery with delayed controlateral orchiectomy were performed in 20 young rats. Only unilateral orchiectomy was planned in 20 other young rats and the 10 remaining were the absolute control group. Mating was observed for 2 weeks after 14 weeks of life. In adult rats, 20 underwent a bilateral ligation and division of the spermatic artery while 20 others were submitted to unilateral ligation-division associated with controlateral orchiectomy in 20 others. The 10 remaining represented the control group. The mating period was 3 weeks. After sacrificing animals, results were noted with regard to histological features and fertility. Among young rats, 45% were fertile and had normal gonadal tissue. From adult rats, only 10 to 15% were fertile. Atrophic testes were observed in 55% of infertile young rats while acute inflammatory lesions were predominant in most of adults. We conclude that ligation or division of spermatic artery is responsible for histological changes thus occurring in infertility in young and adults rats.     

Immunological factors in endometriosis-associated reproductive failure: studies in fertile and infertile women with and without endometriosis

Immunopathophysiological mechanisms in endometriosis-associated reproductive failure were studied in appropriate populations: infertile and fertile women with and without endometriosis. The incidence of sera positive for any of the autoantibodies tested among infertile women with endometriosis (n = 25) was similar to that observed in the three control groups [unexplained infertility patients (n = 25) and fertile women with (n = 10) and without (n = 25) endometriosis]. The mean volume of peritoneal fluid was significantly elevated in women with endometriosis (both fertile and infertile) as compared with patients without endometriosis (fertile or infertile). The concentration of peritoneal fluid leukocytes and the percentage of cells positive for macrophage markers were significantly increased and the percentage of T lymphocytes significantly decreased in infertile women with endometriosis but not in patients with unexplained infertility and fertile women with endometriosis, as compared with fertile controls without endometriosis. Macrophages from infertile patients with endometriosis had higher sperm phagocytosis than did those from infertile women without endometriosis or fertile subjects with or without endometriosis. Incidences of serum and peritoneal fluid samples embryotoxic to the in-vitro development of 2-cell mouse embryos were significantly higher in infertile patients with endometriosis than in unexplained infertility patients and fertile women with or without endometriosis. It is concluded that immunological mechanisms of endometriosis-associated infertility exist but that these peritoneal immunological factors in infertile women with endometriosis are related to their subfertility rather than to the presence of ectopic endometrial implants. This is supported by the lack of immunological abnormalities observed among fertile women with endometriosis. These immunological abnormalities are lacking in patients with unexplained infertility.      

Association of molecular variants of luteinizing hormone with male infertility

Luteinizing hormone (LH) stimulates the interstitial Leydig cells to produce testosterone, which is essential for spermatogenesis. Abnormalities in the function of LH may affect the process of spermatogenesis and thus result in infertility. The aim of this study was to determine the association of three known variants of LH (Gln54Arg [Trp8Arg; Ile15Thr] and Gly102Ser) with male infertility. A total of 145 infertile men and 200 healthy fertile men were recruited and screened for the presence of these three LH variants. The Gln54Arg variant could not be detected in either of the groups studied. Twelve infertile (8.2%) and 15 fertile (7.5%) men were found to carry the [Trp8Ile; I15Thr] variant, but its occurrence did not show any significant difference between the patient and control groups. The Gly102Ser variant was detected in five patients with infertility (3.4%), but not in the control subjects (P = 0.013). This study showed that the Gln54Arg and [Trp8Ile; I15Thr] variants in the LHbeta gene were not associated with male infertility, whereas the Gly102Ser variant might be implicated in infertility in some Singapore Chinese men.     

The effect of an immunoglobulin mu transgene on B cell maturation.

In mu 17.2.25-transgenic (M54) mice the absolute number of surface IgM (sIgM) B cells in lymphoid organs is drastically reduced compared to normal C57BL/6 mice and a high frequency of B cells express the immunoglobulin (Ig) encoded by the transgene rather than endogenous Ig on the surface. To determine the effect of a mu transgene on B cell development, adoptive cell transfers were performed using mu transgenic (M54) bone marrow and fetal liver cells. The data presented support the following conclusions: (a) adult transgenic bone marrow contains functional B cell precursors able to mature and repopulate the spleen and peritoneum of recipient mice. The relative frequency of transgene (sIgMa) and endogenous (sIgMb) surface sIgM-positive B cells reconstituted by transgenic bone marrow in allotype-matched C57BL/6 recipients is the same as in the M54 donors; (b) serum analysis indicates that transgenic bone marrow donor cells can reconstitute B cells in congenic and severe combined immunodeficient (SCID) recipient mice; (c) transgenic fetal liver cells are not a richer source of precursors for B cells expressing endogeneous Ig; (d) in transgenic mice sIgM+ B cells are not restricted to the CD5+ phenotype, however, the relative frequency of sIgMb B cells that are CD5+ is higher in transgenic than normal mice; and (e) bone marrow cells from adult normal and transgenic mice are able to generate CD5+ B lymphocytes in the spleen and peritoneum of allotype-congenic and neonatal SCID recipient mice. The results indicate that the presence of a complete mu heavy chain transgene does not result in a selective developmental block of "conventional" bone marrow-derived pre-B and B cells.     

The differential effect of injecting estradiol-17beta, testosterone, and hydrocortisone during the immune adaptive period on the fertility of female mice

PROBLEM: Female mice injected with estradiol-17beta (E2) and testosterone during the immune adaptive period are infertile as adults. Study 1 examined the effect of the day of injection of E2 and testosterone on the incidence of infertility in two strains of mice. Study 2 examined the effect of hydrocortisone on E2-induced infertility. METHOD OF STUDY: Study 1: Neonatal (C57BL/6J x A/J)F1 B6A and (C3H/HeJ x 129J)F1 C31 female mice were injected from 0 to 3 and from 3 to 6 days of age with either 20 microg E2 or 20 microg testosterone. Animals were tested for fertility by mating with fertile males. Study 2: Neonatal B6A females were injected with 20 microg E2 with/without 1000 microg hydrocortisone on days 1, 3, 5, 7, and 10. At adulthood, ovaries were examined for the presence of corpora lutea (CLs). RESULTS: Study 1: The incidence of E2-induced infertility in adult B6A and C31 females decreased over three consecutive matings. In contrast, the incidence of testosterone-induced infertility in adult B6A and C31 females increased. E2 caused the highest incidence of infertility in C31 females when injected prior to 3 days of age. In B6A mice, E2 caused the highest incidence of infertility when injected after 3 days of age. Study 2: When hydrocortisone was injected with E2, 90% of the B6A females had ovaries with CLs at 100 days of age. Without hydrocortisone, only 16% of the B6A females injected with E2 had ovaries with CLs. CONCLUSION: Study 1: The incidence of infertility caused by injections of E2 is dependent on the strain of mice and the day(s) injected. The incidence of infertility caused by injections of testosterone is independent of the strain of mice. Study 2: Hydrocortisone prevents E2-induced infertility. It is proposed that injections of E2 during the immune adaptive period alter T-cell maturation, which contributes to E2-induced infertility.     

T-Helper 2 Type Cytokine and Soluble Interleukin-2 Receptor Levels in Seminal Plasma of Infertile Men

PROBLEM: The role of cell-mediated immunity (CMI) in unexplained male infertility and impaired sperm function has been explored. METHOD OF STUDY: The presence of cytokines, namely, interleukin-4 (IL-4), interleukin-6 (IL-6), and the soluble interleukin-2 receptor (SIL-2R), was investigated in seminal plasma of 18 fertile and 20 infertile subjects, using specific enzyme-linked immunoadsorbent assays. RESULTS: IL-4 was not detected. SIL-2R was detected, but the concentration difference between the fertile and infertile group was not significant. IL-6 was detected with significantly higher levels in the infertile group compared to the fertile group. IL-6 levels in seminal plasma correlated positively with leukocyte count and negatively with sperm count, motility, and morphology. CONCLUSIONS: These findings show: a) a lack of IL-4 in seminal plasma; b) similar SIL-2R levels in fertile and infertile seminal plasma; c) increased IL-6 secretion in seminal plasma of infertile subjects; and d) specific correlations of IL-6 with the main semen parameters.     

Origin and evolution of somatic cell testicular tumours in transgenic mice

Transgenic mice bearing a construct in which the expression of the SV40 oncogene is directed by the AMH promoter (AT mice) develop testicular tumours in adult life. We aimed to study early steps of tumour development and characterize tumours at different ages by histological, morphometric, and immunohistochemical techniques. One‐ to 3‐month‐old AT mice depicted multifocal Leydig cell hyperplasia. The testicular volume occupied by interstitial tissue was significantly higher in 3‐month‐old AT mice in comparison with littermate controls. Between 5 1/2 and 7 months, microscopic interstitial tumours developed that progressively evolved to form large confluent areas of high mitotic index in 7‐ to 14‐month‐old AT mice. Tumour cells had the characteristics and histoarchitecture of Leydig cells, or formed solid cord‐like structures reminiscent of those seen in Sertoli cell tumours. Hyperplastic areas and tumours diffusely expressed 3β‐hydroxysteroid dehydrogenase (3β‐HSD) in Leydig cell areas. AMH expression was negative in Leydig cell conglomerates and tumours and variable in cord‐like tumours. The SV40 T antigen and markers of cell proliferation (PCNA) were intensely positive in hyperplastic cells and tumours. Control mice of similar ages showed neither hyperplasia nor tumours, and SV40 T expression was always negative. In conclusion, transgenic mice develop large testicular tumours that are preceded by interstitial hyperplasia and microtumours. The histological and immunohistochemical phenotype of tumours (Leydig and Sertoli cell differentiation, positive 3β‐HSD, and variable AMH) suggests a mixed differentiation of somatic cells of the specialized gonadal stroma. The finding that an oncogene directed by a promoter specifically active in fetal Sertoli cells has given rise to testicular tumours of mixed differentiation is compatible with a common origin of Leydig and Sertoli cells from the specific stroma of the gonadal ridge, as supported by double labelling experiments in fetal mice showing co‐localization of the transgene with Sertoli and Leydig cell markers. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Smad2和Smad4在家猫睾丸发育和精子发生中的表达与定位

目的 研究转化生长因子β(TGF-β)超家族的下游信号转导分子Smad2和Smad4蛋白,在不同发育阶段家猫睾丸中的表达和定位,探索Smad2和Smad4蛋白与家猫睾丸发育和精子发生的关系. 方法 应用免疫组织化学技术,研究Smad2和Smad4蛋白在幼年(n=3)、青春期(n=3)和性成熟(n=18)睾丸中的定位,并通过Western blotting技术对免疫组织化学中所用抗体的特异性进行了检测. 结果 免疫组织化学结果显示,Smad2和Smad4蛋白定位于各发育阶段家猫睾丸的生殖细胞、支持细胞和间质细胞的胞质中;Western blotting结果显示,多克隆兔抗Smad2和Smad4抗体与家猫睾丸蛋白提取物中分子量约为58kD、66kD的蛋白条带发生免疫阳性反应. 结论 Smad2和Smad4蛋白在家猫睾丸发育和精子发生的各个阶段均有表达,提示其参与睾丸发育和精子发生的调节.     

Healing of Burn Wounds in Transgenic Mice Overexpressing Transforming Growth Factor-β1 in the Epidermis

Transforming growth factor-beta (TGF-beta) isoforms are multifunctional cytokines that play an important role in wound healing. Transgenic mice overexpressing TGF-beta in the skin under control of epidermal-specific promoters have provided models to study the effects of increased TGF-beta on epidermal cell growth and cutaneous wound repair. To date, most of these studies used transgenic mice that overexpress active TGF-beta in the skin by modulating the latency-associated-peptide to prevent its association with active TGF-beta. The present study is the first to use transgenic mice that overexpress the natural form of latent TGF-beta 1 in the epidermis, driven by the keratin 14 gene promoter to investigate the effects of locally elevated TGF-beta 1 on the healing of partial-thickness burn wounds made on the back of the mice using a CO(2) laser. Using this model, we demonstrated activation of latent TGF-beta after wounding and determined the phenotypes of burn wound healing. We found that introduction of the latent TGF-beta1 gene into keratinocytes markedly increases the release and activation of TGF-beta after burn injury. Elevated local TGF-beta significantly inhibited wound re-epithelialization in heterozygous (42% closed versus 92% in controls, P < 0.05) and homozygous (25% versus 92%, P < 0.01) animals at day 12 after wounding. Interestingly, expression of type I collagen mRNA and hydroxyproline significantly increased in the wounds of transgenic mice, probably as a result of a paracrine effect of the transgene.     

Localization of intestinal intraepithelial T lymphocytes involves regulation of alphaEbeta7 expression by transforming growth factor-beta

Induction of alphaEbeta7 expression on T cells by transforming growth factor (TGF)-beta is thought to be important for intestinal intraepithelial T lymphocyte (IEL) entry into the epithelial compartment. However, there has been no in vivo evidence that up-regulation of alphaEbeta7 expression on T cells by TGF-beta is critical for the selective localization of intestinal IEL in the epithelial area. We have recently established transgenic mice expressing Smad7 under the control of a distal lck promoter where TGF-beta/Smad signaling is specifically blocked in mature T cells. Here we showed that TGF-beta-mediated up-regulation of alphaEbeta7 was impaired on T cells isolated from the Smad7 transgenic mice associated with reduced numbers of intestinal IEL when compared with that in wild-type littermates. These results indicated that failure to induce alphaEbeta7 on T cells by TGF-beta resulted in reduced numbers of intestinal IEL, suggesting the importance of alphaEbeta7 expression by TGF-beta in selective localization of intestinal IEL.