AN INVESTIGATION INTO THE MOLECULAR MECHANISMS OF ANDROGEN ACTION IN THE SERTOLI CELL

Steroid hormones regulate target cell function via quantitative and qualitative changes in RNA and protein synthesis. In the testis, androgens are known to play an important role in the regulation of spermatogenesis. The Sertoli cell (SC), whose function is thought to be supportive to the developing germ cell, has been implicated as an androgen target cell. Although cytoplasmic androgen receptors and chromatin acceptor sites for androgen-receptor complexes have been found in SC, effects on RNA synthesis have not previously been demonstrated. In this study, SC RNA synthetic activity was characterized and the effect of testosterone on SC nuclear transcriptional activity in vitro assessed. SC exhibited two fold increases in RNA and ribonucleotide pool concentrations during sexual maturation. These changes appeared to correlate with a previously observed increase in protein concentration per cell over an age span of 15-60 days. Following incubation with ('3)H-uridine, SC from older animals incorporated more label into RNA than SC from younger animals. Since the relative concentration of cytidine nucleotides was higher in SC from older rats, the age-related increase in tritium incorporation may reflect an associated increase in incorporation of ('3)H-CMP into RNA. Alternatively, the enhanced labeling may be the result of either a change in the base composition of the RNA resulting in a higher proportion of CMP and UMP in the RNA, or compartmentalization of the nucleotide pools. The physiologic consequences of these maturational alterations of nucleotide pools remains to be elucidated. RNA polymerase activities were characterized in intact nuclei obtained from cultured rat SC. (alpha)-Amanitin resistant RNA polymerase I+III activity was identical when measured in low or high ionic strength (0.05 M or 0.25 M ammonium sulfate (AS)) in the presence of MnCl(,2) or MgCl(,2), with a divalent cation optimum of 1.6 mM. RNA polymerase II was most active in 0.25 M AS and 1.6 mM MnCl(,2). The apparent Km of RNA polymerase II for UTP was 0.016 mM in 0.05 M AS and 0.037 mM in 0.25 M AS. The apparent Km values for total polymerase activity was 0.008 mM and 0.036 mM at low and high ionic strenghts, respectively. These data indicate that Sertoli cell RNA polymerase activities have catalytic properties characteristic of eukaryotic polymerase activities in general. In the presence of 21 (mu)M testosterone, RNA polymerase II activity increased two fold at 15 minutes, then declined but was still elevated over control values six hours after androgen addition. Polymerase I+III activity was not greatly affected by testosterone. The stimulation of polymerase II measured at 15 minutes was dose-dependent, with a maximum at 0.53 nM and no further stimulation up to 10('-5) M (ED(,50) = 0.25 nM testosterone), and was androgen specific. The results of preliminary RNA isolation and characterization experiments suggested that the synthesis of several species of RNA was enhanced by testosterone administration. These findings have great potential importance since they represent the first demonstration of a direct effect of androgens on the transcriptional process in the Sertoli cell. Furthermore, the results of these studies constitute further evidence that the Sertoli cell is a target for androgen action in the testis. ^

GENETIC INTERACTIONS OF FACTORS THAT REGULATE ALTERNATIVE RNA SPLICING IN THE MALE GERM LINE OF DROSOPHILA

Alternative RNA splicing is a critical process that contributes variety to protein functions, and further controls cell differentiation and normal development. Although it is known that most eukaryotic genes produce multiple transcripts in which splice site selection is regulated, how RNA binding proteins cooperate to activate and repress specific splice sites is still poorly understood. In addition how the regulation of alternative splicing affects germ cell development is also not well known. In this study, Drosophila Transformer 2 (Tra2) was used as a model to explore both the mechanism of its repressive function on its own pre-mRNA splicing, and the effect of the splicing regulation on spermatogenesis in testis. Half-pint (Hfp), a protein known as splicing activator, was identified in an S2 cell-based RNAi screen as a co-repressor that functions in combination with Tra2 in the splicing repression of the M1 intron. Its repressive splicing function is found to be sequence specific and is dependent on both the weak 3’ splice site and an intronic splicing silencer within the M1 intron. In addition we found that in vivo, two forms of Hfp are expressed in a cell type specific manner. These alternative forms differ at their amino terminus affecting the presence of a region with four RS dipeptides. Using assays in Drosophila S2 cells, we determined that the alternative N terminal domain is necessary in repression. This difference is probably due to differential localization of the two isoforms in the nucleus and cytoplasm. Our in vivo studies show that both Hfp and Tra2 are required for normal spermatogenesis and cooperate in repression of M1 splicing in spermatocytes. But interestingly, Tra2 and Hfp antagonize each other’s function in regulating germline specific alternative splicing of Taf1 (TBP associated factor 1). Genetic and cytological studies showed that mutants of Hfp and Taf1 both cause similar defects in meiosis and spermatogenesis. These results suggest Hfp regulates normal spermatogenesis partially through the regulation of taf1 splicing. These observations indicate that Hfp regulates tra2 and taf1 activity and play an important role in germ cell differentiation of male flies.

Highly efficient base excision repair (BER) in human and rat male germ cells

The quality of germ cell DNA is critical for the fate of the offspring, yet there is limited knowledge of the DNA repair capabilities of such cells. One of the main DNA repair pathways is base excision repair (BER) which is initiated by DNA glycosylases that excise damaged bases, followed by incision of the generated abasic (AP) sites. We have studied human and rat methylpurine-DNA glycosylase (MPG), uracil-DNA glycosylase (UNG), and the major AP endonuclease (HAP1/APEX) in male germ cells. Enzymatic activities and western analyses indicate that these enzymes are present in human and rat male germ cells in amounts that are at least as high as in somatic cells. Minor differences were observed between different cellular stages of rat spermatogenesis and spermiogenesis. Repair of methylated DNA was also studied at the cellular level using the Comet assay. The repair was highly efficient in both human and rat male germ cells, in primary spermatocytes as well as round spermatids, compared to rat mononuclear blood cells or hepatocytes. This efficient BER removes frequently occurring DNA lesions that arise spontaneously or via environmental agents, thereby minimising the number of potential mutations transferred to the next generation.

Induction of primordial germ cells from murine epiblasts by synergistic action of BMP4 and BMP8B signaling pathways

Extraembryonic ectoderm-derived factors instruct the pluripotent epiblast cells to develop toward a restricted primordial germ cell (PGC) fate during murine gastrulation. Genes encoding Bmp4 of the Dpp class and Bmp8b of the 60A class are expressed in the extraembryonic ectoderm and targeted mutation of either results in severe defects in PGC formation. It has been shown that heterodimers of DPP and 60A classes of bone morphogenetic proteins (BMPs) are more potent than each homodimers in bone and mesoderm induction in vitro, suggesting that BMP4 and BMP8B may form heterodimers to induce PGCs. To investigate how BMP4 and BMP8B interact and signal for PGC induction, we cocultured epiblasts of embryonic day 6.0–6.25 embryos with BMP4 and BMP8B proteins produced by COS cells. Our data show that BMP4 or BMP8B homodimers alone cannot induce PGCs whereas they can in combination, providing evidence that two BMP pathways are simultaneously required for the generation of a given cell type in mammals and also providing a prototype method for PGC induction in vitro. Furthermore, the PGC defects of Bmp8b mutants can be rescued by BMP8B homodimers whereas BMP4 homodimers cannot mitigate the PGC defects of Bmp4 null mutants, suggesting that BMP4 proteins are also required for epiblast cells to gain germ-line competency before the synergistic action of BMP4 and BMP8B.

Postembryonic segregation of the germ line in sea urchins in relation to indirect development.

The four small micromeres of the sea urchin embryo contribute only to the coelomic sacs, which produce major components of the adult body plan during postembryonic development. To test the proposition that the small micromeres are the definitive primordial germ cell lineage of the sea urchin, we deleted their 4th cleavage parents, and raised the deleted embryos through larval life and metamorphosis to sexual maturity. Almost all of the experimental animals produced functional gametes, excluding the possibility that the germ cell lineage arises exclusively and obligatorily from descendants of the small micromeres; rather, the germ cell lineage arises during the postembryonic development of the rudiment. A survey of the literature indicates that there is no known case of an embryonic primordial germ cell lineage in a bilaterian species that displays maximal indirect development.

Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice.

The lacZ transgenic mouse (Muta mouse) model was used to examine the timing of ethylnitrosourea (ENU)-induced mutations in germ cells. The spectrum of mutations was also determined. Animals received five daily treatments with ENU at 50 mg/kg and were sampled at times up to 55 days after treatment. In mixed germ-cell populations isolated from seminiferous tubules, there was little increase in the mutant frequency 5 days after treatment; subsequently, there was a continuous increase until the maximum (17.5-fold above background) was reached by approximately 35 days. In the spermatozoa, an increase in mutant frequency was not seen until 20 days after treatment, with the maximum (4.3-fold above background) being achieved no sooner than approximately 35 days. Based on the timing of sampling, these data demonstrate the detection of both spermatogonial and postspermatogonial, mutations. The most prominent feature of the ENU-induced base-pair mutations in testicular germ cells sampled 55 days after treatment is that 70% are induced in A.T base pairs, compared to only 16% in spontaneous mutations. These findings are consistent with comparable data from ENU studies using assays for inherited germ-cell mutations in mice. This study has demonstrated the utility and potential of the transgenic mouse lacZ model (Muta mouse) for the detection and study of germ-cell mutations and provides guidance in the selection of simplified treatment and sampling protocols.

Germ cells enter meiosis in a rostro-caudal wave during development of the mouse ovary

Germ cells in the mouse embryo remain undifferentiated until about 13.5 days post-coitum (dpc), when male germ cells enter mitotic arrest and female germ cells enter meiosis. The molecular signals and transcriptional control mechanisms governing the differential fate of germ cells in males and females remain largely unknown. In order to gain insights into the behavior of germ cells around this period and into likely mechanisms controlling entry into meiosis, we have studied by wholemount in situ hybridization the expression pattern of two germ cell-specific markers, Oct4 and Sycp3, during mouse fetal gonad development. We observed a dynamic wave of expression of both genes in developing ovaries, with Oct4 expression being extinguished in a rostro-caudal wave and Sycp3 being upregulated in a corresponding wave, during the period 13.5-15.5 dpc. These results indicate that entry into meiosis proceeds in a rostro-caudal progression, in turn suggesting that somatically derived signals may contribute to the control of germ cell entry into meiosis in developing ovaries. (C) 2004 Wiley-Liss, Inc.

HMG box transcription factor gene Hbp1 is expressed in germ cells of the developing mouse testis

HMG box containing protein 1 (HBP1) is a high mobility group domain transcriptional repressor that regulates proliferation in differentiated tissues. We have found mouse Hbp1 to be expressed strongly in the embryonic mouse testis from approximately 12.5 days post coitum, compared with low levels of expression in the embryonic ovary. Expression of Hbp1 is maintained in the developing testis beyond the onset of spermatogenesis after birth. Whole-mount in situ hybridisation analysis showed that expression of Hbp1 in the XY gonad is localized within the developing testis cords, the precursors of the seminiferous tubules. Expression of Hbp1 is not apparent in testis cords of gonads from homozygous We mutant embryos, which lack germ cells. In situ hybridisation analysis on cryosectioned embryonic testis indicated that Hbp1 expression resembles that of the germ cell marker Oct4. We conclude that Hbp1 is up-regulated specifically in germ cells of the developing XY gonad. The expression of Hbp1 in XY germ cells appears to correlate with the onset of mitotic arrest in these cells. (C) 2004 Wiley-Liss, Inc.

NOTCH1 Gain of Function in Germ Cells Causes Failure of Spermatogenesis in Male Mice

NOTCH1 is a member of the NOTCH receptor family, a group of single-pass trans-membrane receptors. NOTCH signaling is highly conserved in evolution and mediates communication between adjacent cells. NOTCH receptors have been implicated in cell fate determination, as well as maintenance and differentiation of stem cells. In the mammalian testis expression of NOTCH1 in somatic and germ cells has been demonstrated, however its role in spermatogenesis was not clear. To study the significance of NOTCH1 in germ cells, we applied a cre/loxP approach in mice to induce NOTCH1 gain- or loss-of function specifically in male germ cells. Using a Stra8-icretransgene we produced mice with conditional activation of the NOTCH1 intracellular domain (NICD) in germ cells. Spermatogenesis in these mutants was progressively affected with age, resulting in decreased testis weight and sperm count. Analysis of downstream target genes of NOTCH1 signaling showed an increased expression of Hes5, with a reduction of the spermatogonial differentiation marker, Neurog3 expression in the mutant testis. Apoptosis was significantly increased in mouse germ cells with the corresponding elevation of pro-apoptotic Trp53 and Trp63genes' expression. We also showed that the conditional germ cell-specific ablation of Notch1 had no effect on spermatogenesis or male fertility. Our data suggest the importance of NOTCH signaling regulation in male germ cells for their survival and differentiation.

Ovarian sertoli-leydig cell tumour with heterologous elements of gastrointestinal type associated with elevated serum alpha-fetoprotein level: an unusual case and literature review

Here we describe the case of a 19-year-old woman with a poorly differentiated ovarian Sertoli-Leydig cell tumor and an elevated serum alphafetoprotein level. The patient presented with diffuse abdominal pain and bloating. Physical examination, ultrasound, and magnetic resonance imaging revealed a right ovarian tumor that was histopathologically diagnosed as a poorly differentiated Sertoli-Leydig cell tumor with heterologous elements. Her alpha-fetoprotein serum level was undetectable after tumor resection. Sertoli-Leydig cell tumors are rare sex cord-stromal tumors that account for 0.5% of all ovarian neoplasms. Sertoli-Leydig cell tumors tend to be unilateral and occur in women under 30 years of age. Although they are the most common virilizing tumor of the ovary, about 60% are endocrineinactive tumors. Elevated serum levels of alpha-fetoprotein are rarely associated with Sertoli-Leydig cell tumors, with only approximately 30 such cases previously reported in the literature. The differential diagnosis should include common alpha-fetoprotein-producing ovarian entities such as germ cell tumors, as well as other non-germ cell tumors that have been rarely reported to produce this tumor marker.

Homozygous Inactivating Mutation In Nanos3 In Two Sisters With Primary Ovarian Insufficiency.

Despite the increasing understanding of female reproduction, the molecular diagnosis of primary ovarian insufficiency (POI) is seldom obtained. The RNA-binding protein NANOS3 poses as an interesting candidate gene for POI since members of the Nanos family have an evolutionarily conserved function in germ cell development and maintenance by repressing apoptosis. We performed mutational analysis of NANOS3 in a cohort of 85 Brazilian women with familial or isolated POI, presenting with primary or secondary amenorrhea, and in ethnically-matched control women. A homozygous p.Glu120Lys mutation in NANOS3 was identified in two sisters with primary amenorrhea. The substituted amino acid is located within the second C2HC motif in the conserved zinc finger domain of NANOS3 and in silico molecular modelling suggests destabilization of protein-RNA interaction. In vitro analyses of apoptosis through flow cytometry and confocal microscopy show that NANOS3 capacity to prevent apoptosis was impaired by this mutation. The identification of an inactivating missense mutation in NANOS3 suggests a mechanism for POI involving increased primordial germ cells (PGCs) apoptosis during embryonic cell migration and highlights the importance of NANOS proteins in human ovarian biology.

[preservation Of The Fertility And The Ovaries In Women With Benign Adnexal Tumors].

To evaluate the sparing of fertility and ovaries in women submitted to surgical treatment for benign adnexal tumors. Between February 2010 and January 2014, 206 patients were included in this observational study as they were submitted to surgical treatment for benign ovarian tumors at CAISM, a tertiary hospital. Fertility sparing surgery was defined as tumorectomy or unilateral salpingoophorectomy without hysterectomy in premenopausal women. Preservation of the ovary occurred when at least one ovary or part of it was mantained. Of the 206 women with benign tumors, 120 (58%) were premenopausal and 86 (42%) were postmenopausal. There were 36 (30%) ovarian germ cell tumors, 31 (26%) epithelial neoplasms and 11 (9%) sex-cord stromal tumors among premenopausal women. In the group of postmenopausal women, 35 (41%) epithelial neoplasms, 27 (31%) sex-cord stromal tumors and 8 (9%) ovarian germ cell tumors were identified. Among 36 women with non-neoplastic ovarian tumors, 21 (58%) had endometriomas and 8 (22%) functional cysts. Among 22 women with extra-ovarian tumors, uterine leiomyomatosis was the most frequent finding (50%). In the group of women who were ≤ 35 years old, 26 (57%) were treated by tumorectomy and 18 (39%) were submitted to unilateral salpingoophorectomy with sparing of the uterus and the contralateral ovary. Women who were ≤ 35 years old were more frequently operated by laparoscopy which was associated with a higher number of fertility sparing procedures when compared to laparotomy (p<0.01). Twenty-six (28%) women submitted to hysterectomy with bilateral salpingoophorectomy were premenopausal. Although there is a trend to perform only tumorectomy in women who are ≤ 35 years old, a significant number of young women is still treated by salpingoophorectomy. Among 36- to 45-year-old women, only 70% had their fertility spared, while 20% had both ovaries removed. However, whenever possible, we must try to preserve the ovaries, mainly in premenopausal women.

Cisplatin-based therapy: A neurological and neuropsychological review

The present paper reviews research in the area of the broad-spectrum chemotherapeutic agent cisplatin (cis-diamminedichloro-platinum II) and examines the implications for clinical neuropsychology arising from the neurological disruption associated with cisplatin-based therapy. The paper begins with a brief review of cisplatin treatment in terms other than survival alone, and examines the side-effects and the potential central nervous system (CNS) dysfunction in terms of neurological symptoms and concomitant implications for neuropsychology. Two main implications for clinical neuropsychology arising from cisplatin therapy are identified. First, cisplatin therapy impacts upon the psychological well-being of the patient, particularly during and in the months following treatment. It is suggested that during this time, a primary role for neuropsychology is to focus upon the monitoring and the active enhancement of the patient's social, psychological and spiritual resources. Second, with regard to neurocognitive changes, the review suggests that (1) neurocognitive assessment may not yield stable results within 8 months following treatment and (2) while perceptual, memory, attentional and executive dysfunction may be predicted following cisplatin treatment, little systematic research has been carried out to investigate such a possibility. Future research might profitably address this issue and also specifically examine the effects of low dosage cisplatin-based therapy and the effects of recently developed neuroprotective agents. Finally, there is some evidence to suggest that women may be more susceptible to neurotoxicity during cisplatin therapy, but no gender-related cognitive effects are reported in the cisplatin literature. Future research could usefully investigate gender differences in association with cisplatin chemotherapy. Copyright (C) 2000 John Wiley & Sons, Ltd.

Early onset of primary hypogonadism revealed by serum anti-Mullerian hormone determination during infancy and childhood in trisomy 21

Male patients with an extra sex chromosome or autosome are expected to present primary hypogonadism at puberty owing to meiotic germ-cell failure. Scarce information is available on trisomy 21, a frequent autosomal aneuploidy. Our objective was to assess whether trisomy 21 presents with pubertal-onset, germ-cell specific, primary hypogonadism in males, or whether the hypogonadism is established earlier and affects other testicular cell populations. We assessed the functional status of the pituitary-testicular axis, especially Sertoli cell function, in 117 boys with trisomy 21 (ages: 2 months-20 year). To compare with an adequate control population, we established reference levels for serum anti-Mullerian hormone (AMH) in 421 normal males, from birth to adulthood, using a recently developed ultrasensitive assay. In trisomy 21, AMH was lower than normal, indicating Sertoli cell dysfunction, from early infancy, independently of the existence of cryptorchidism. The overall prevalence rate of AMH below the 3rd percentile was 64.3% in infants with trisomy 21. Follicle-stimulating hormone was elevated in patients <6 months and after pubertal onset. Testosterone was within the normal range, but luteinizing hormone was elevated in most patients <6 months and after pubertal onset, indicating a mild Leydig cell dysfunction. We conclude that in trisomy 21, primary hypogonadism involves a combined dysfunction of Sertoli and Leydig cells, which can be observed independently of cryptorchidism soon after birth, thus prompting the search for new hypotheses to explain the pathophysiology of gonadal dysfunction in autosomal trisomy.

Evaluation of Efficacy and Safety of Zinc Gluconate Associated with Dimethyl Sulphoxide for Sexually Mature Canine Males Chemical Neutering

Contents The aim of this research was to evaluate the efficacy of zinc gluconate associated with dimethyl sulphoxide (DMSO) for chemical neutering in canine males. Fifteen sexually mature male dogs were divided in two groups, named control and treated. An injection was administered to both testicles, at a concentration of 26.2 mg zinc gluconate per ml and 0.5% DMSO in the treated group (11 dogs). The control group was given injections of saline solution (four dogs). Clinical examination and blood collection for a haemogram were done both before and after drug injection. There were 12 spermograms performed to analyse sperm motility, sperm vigour, ejaculate volume, testicle size, pathology and sperm concentrations. Libido was also measured. An ultrasound examination and histopathology were performed at the end of the experiment. Dogs` libido after chemical injection was reduced by over 50%. The spermogram analysis showed final mean results of 14.54% for sperm motility, 0.72 of sperm vigour and 37 150 per million spermatozoa per millilitre, values considered below the necessary levels at which fertilization can occur. Ultrasound and histopathology analyses of testicles for the treated group revealed more intense injuries when compared with the control group, with compromised testicular parenchyma and a decrease of germ cell number leading to total atrophy, indicating that the treatment reduced the fertilizing potential of male dogs, promoting a possible subfertile status.