Isolation of bovine herpesvirus type 5 from the semen of a healthy bull in Australia.

Artificial insemination is widely used in the cattle industry and a major challenge is to ensure that semen is free of infectious agents. A healthy donor bull was tested for freedom from infectious agents. A bovine herpesvirus was isolated in testis cells and identified as bovine herpesvirus type 5 (BoHV-5) by polymerase chain reaction and by direct amplicon sequencing. The amplicon sequence shared 100% similarity with the published sequence of BoHV-5. This is the first report in Australia of BoHV-5 in semen. The implications of this finding are discussed.

Stem Cell Markers in Cancer: Do They Have Clinical Significance?

In cancer, a subpopulation of malignant cells expresses markers of normal stem cells. These cells have the potential of initiating tumor growth and therefore also tumor recurrence. Thus, these cells are called cancer stem cells. A myriad of markers have been applied to identify these cells, but no single marker can be found exclusively in cancer stem cells. In many types of cancer, clinical recurrence and tumor progression are the main causes of mortality, despite intense oncological treatment. It has been proposed that the presence of cancer stem cells causes this resistance to therapy. The scope of this thesis is to investigate the role of stem cell markers and genes in the clinical setting. Especially, the aim was to elucidate the clinical significance of stem cell markers as novel prognostic and diagnostic tools in cancer. Tumor biopsy material from central nervous system tumors (oligodendroglioma, astrocytoma and glioblatoma), neural crest derived tumors (pheochromocytomas) and oral carcinoma was screened for stem cell markers. Initially, 15 stem cell markers were screened in a test series of gliomas. The markers applied for expanded tumor analyses (in 305 cases of glioma, 42 cases of pheochromocytoma, and 73 cases of oral carcinoma) were BMI-1, Snail, p16, mdm2, and c-Myc. Data on marker expression was compared with clinical and pathological parameters. In gliomas, BMI-1 expression was found in nearly all tumors analyzed, but the frequency of BMI-1 expressing cells was highly variable, ranging from 1 to 100%. In oligodendroglioma, BMI-1 expression was identified as a prognostic marker independent of tumor grade and clinical parameters. In pheochromocytoma, Snail expression was shown to distinguish between the metastatic and non-metastatic forms of the tumor. Snail expression was seen only in metastatic tumors, whereas non-metastatic tumors did not commonly express Snail. Finally, in oral carcinoma, BMI-1 expression was seen in roughly 80% of tumors, and Snail expression was high or very high in all cases. The lack of BMI-1 expression was associated with early relapse in oral carcinoma.

Androgen receptor-dependent and independent functions of ARIP4

Androgen receptor (AR) is necessary for normal male phenotype development and essential for spermatogenesis. AR is a classical steroid receptor mediating actions of male sex steroids testosterone and 5-alpha-dihydrotestosterone. Numerous coregulators interact with the receptor and regulate AR activity on target genes. This study deals with the characterization of androgen receptor-interacting protein 4 (ARIP4). ARIP4 binds DNA, interacts with AR in vitro and in cultured yeast and mammalian cells, and modulates AR-dependent transactivation. ARIP4 is an active DNA-dependent ATPase, and this enzymatic activity is essential for the ability of ARIP4 to modulate AR function. On the basis of sequence homology in its ATPase domain, ARIP4 belongs to the SNF2 family of proteins involved in chromatin remodeling, DNA repair, and homologous recombination. Similar to its closest homologs ATRX and Rad54, ARIP4 does not seem to be a classical chromatin remodeling protein in that it does not appear to form large protein complexes in vivo or remodel mononucleosomes in vitro. However, ARIP4 is able to generate superhelical torsion on linear DNA fragments. ARIP4 is covalently modified by SUMO-1, and mutation of six potential SUMO attachment sites abolishes the ability of ARIP4 to bind DNA, hydrolyze ATP, and activate AR function. ARIP4 expression starts in early embryonic development. In mouse embryo ARIP4 is present mainly in the neural tube and limb buds. In adult mouse tissues ARIP4 expression is virtually ubiquitous. In mouse testis ARIP4 is expressed in the nuclei of Sertoli cells in a stage-dependent manner. ARIP4 is also present in the nuclei of Leydig cells, spermatogonia, pachytene and diplotene spermatocytes. Testicular expression pattern of ARIP4 does not differ significantly in wild-type, FSHRKO, and LuRKO mice. In the testis of hpg mice, ARIP4 is found mainly in interstitial cells and has very low, if any, expression in Sertoli and germ cells. Heterozygous Arip4+/ mice are fertile and appear normal; however, they are haploinsufficient with regard to androgen action in Sertoli cells. In contrast, Arip4 / embryos are not viable. They have significantly reduced body size at E9.5 and die by E11.5. Compared to wild-type littermates, Arip4 / embryos possess a higher percentage of apoptotic cells at E9.5 and E10.5. Fibroblasts derived from Arip4 / embryos cease growing after 2-3 passages and exhibit a significantly increased apoptosis and decreased proliferation rate than cells from wild-type embryos. Our findings demonstrate that ARIP4 plays an essential role in mouse embryonic development. In addition, testicular expression and AR coregulatory activity of ARIP4 suggest a role of ARIP4-AR interaction in the somatic cells of the testis.

The Functions and Regulation of Ornithine Decarboxylase

Ornithine decarboxylase (ODC) regulates the synthesis of polyamines which are involved in many cellular functions e.g. proliferation and differentiation. Due to its critical role, ODC is a tightly regulated enzyme by antizymes and antizyme inhibitors. If the regulation fails, the activity of ODC increases and may lead to malignant transformation of a cell. Increased ODC activity is found in many common cancers, including colon, prostate, and breast cancer. In a transformed cell, dynamics of the actin cytoskeleton is disturbed. A small G-protein, RhoA regulates organization of the cytoskeleton, and its overactivity increases malignant potential of the cell. The present results indicate that covalent attachment of polyamines by transglutaminase is a physiological means of regulating the activity of RhoA. The translocation of RhoA to the plasma membrane, where it exerts its activity is dependent on the presence of catalytically active ODC. As the overactivity of ODC and RhoA are implicated in cell transformation, the results provide a mechanistic explanation of the interrelationship between the polyamine metabolism and the reorganization of the actin cytoskeleton occurring in cancer cells. ODC and polyamines have also an important role in the function of central nervous system. They participate in the regulation of brain morphogenesis in embryos. In adult nervous tissue, polyamines regulate K+ and glutamate channels. K+ inward rectifying channels control membrane potentials and NMDA-type glutamate receptors (NMDAR) regulate synaptic plasticity. High ODC activity and polyamine levels are considered important in the development of ischemic brain damage and they are implicated in the pathogenesis of Alzheimer s disease (AD). A homolog of ODC was cloned from a human brain cDNA library, and several alternatively spliced variants were detected in human brain and testis. The novel protein was nevertheless devoid of ODC catalytic activity. It was subsequently found to be a novel inductor of ODC activity and polyamine synthesis, called antizyme inhibitor 2 (AZIN2). The accumulation of AZIN2 in vesicle-like formations along the axons and beneath the plasma membrane of neurons as well as in steroid hormone producing Leydig cells and luteal cells of the gonads implies that AZIN2 plays a role in secretion and vesicle trafficking. An accumulation of AZIN2 was detected also in specimens of AD brains. This increased expression of AZIN2 was specific for AD and was not found in brains with other neurodegenerative diseases including CADASIL or dementia with Lewy bodies.

Novel conditional mouse models for targeted kidney research : Emphasis on the analysis of the biological function of nephrin

Nephrin is a transmembrane protein belonging to the immunoglobulin superfamily and is expressed primarily in the podocytes, which are highly differentiated epithelial cells needed for primary urine formation in the kidney. Mutations leading to nephrin loss abrogate podocyte morphology, and result in massive protein loss into urine and consequent early death in humans carrying specific mutations in this gene. The disease phenotype is closely replicated in respective mouse models. The purpose of this thesis was to generate novel inducible mouse-lines, which allow targeted gene deletion in a time and tissue-specific manner. A proof of principle model for succesful gene therapy for this disease was generated, which allowed podocyte specific transgene replacement to rescue gene deficient mice from perinatal lethality. Furthermore, the phenotypic consequences of nephrin restoration in the kidney and nephrin deficiency in the testis, brain and pancreas in rescued mice were investigated. A novel podocyte-specific construct was achieved by using standard cloning techniques to provide an inducible tool for in vitro and in vivo gene targeting. Using modified constructs and microinjection procedures two novel transgenic mouse-lines were generated. First, a mouse-line with doxycycline inducible expression of Cre recombinase that allows podocyte-specific gene deletion was generated. Second, a mouse-line with doxycycline inducible expression of rat nephrin, which allows podocyte-specific nephrin over-expression was made. Furthermore, it was possible to rescue nephrin deficient mice from perinatal lethality by cross-breeding them with a mouse-line with inducible rat nephrin expression that restored the missing endogenous nephrin only in the kidney after doxycycline treatment. The rescued mice were smaller, infertile, showed genital malformations and developed distinct histological abnormalities in the kidney with an altered molecular composition of the podocytes. Histological changes were also found in the testis, cerebellum and pancreas. The expression of another molecule with limited tissue expression, densin, was localized to the plasma membranes of Sertoli cells in the testis by immunofluorescence staining. Densin may be an essential adherens junction protein between Sertoli cells and developing germ cells and these junctions share similar protein assembly with kidney podocytes. This single, binary conditional construct serves as a cost- and time-efficient tool to increase the understanding of podocyte-specific key proteins in health and disease. The results verified a tightly controlled inducible podocyte-specific transgene expression in vitro and in vivo as expected. These novel mouse-lines with doxycycline inducible Cre recombinase and with rat nephrin expression will be useful for conditional gene targeting of essential podocyte proteins and to study in detail their functions in the adult mice. This is important for future diagnostic and pharmacologic development platforms.

Effect of deprivation of vitamin A on the basic proteins of the nuclei of rat testes

The relative concentrations of the major histones, namely, H1, TH1, H2A, H2B, H3 and H4 are not significantly changed in the testes of the vitamin A-deficient rats, as compared to those in the normal controls. But the testis-specific protein, TP, which is synthesized at the mid-spermatid stage, is markedly reduced in the testes of the deficient rats. On supplementation of the deficient rats with retinyl acetate for 28 days, there was a 50% recovery in the relative concentration of the TP with respect to the total amounts of H1 and TH1.

Molecular Genetics of Lactase Deficiencies

Congenital lactase deficiency (CLD) (MIM 223000) is a rare autosomal recessive gastrointestinal disorder characterized by watery diarrhea in infants fed with breast milk or other lactose-containing formulas. The CLD locus was previously assigned by linkage and linkage disequilibrium analyses on 2q21 in 19 Finnish families. In this study, the molecular background of this disorder is reported. The CLD locus was refined in 32 CLD patients in 24 families by using microsatellite and single nucleotide polymorphism (SNP) haplotypes. Mutation analyses were performed by direct sequencing. We identified 5 distinct mutations in the lactase (LCT) gene, encoding the enzyme that hydrolyzes lactose in the intestinal lumen. These findings facilitate genetic testing of CLD in clinical practice and enable genetic counseling. The present data also provide the basis for detailed characterization of the molecular pathogenesis of this disorder. Adult-type hypolactasia (MIM 223100) (lactase non-persistence, lactose intolerance) is an autosomal recessive gastrointestinal condition that is a result of a decline in the activity of lactase in the intestinal lumen after weaning. Adult-type hypolactasia is considered to be a normal phenomenon among mammals and symptoms are remarkably milder than experienced in CLD. Recently, a variant C/T-13910 was shown to associate with the adult-type hypolactasia trait, locating 13.9 kb upstream of the LCT gene. In this study, the functional significance of the C/T-13910 variant was determined by studying the LCT mRNA levels in intestinal biopsy samples in children and adults with different genotypes. RT-PCR followed by solid-phase minisequencing was applied to determine the relative expression levels of the LCT alleles using an informative SNP located in exon 1. In children, the C-13910 allele was observed to be downregulated after five years of age in parallel with lactase enzyme activity. The expression of the LCT mRNA in the intestinal mucosa in individuals with the T-13910 A-22018 alleles was 11.5 times higher than that found in individuals with the C-13910, G-22018 alleles. These findings suggest that the C/T-13910 associated with adult-type hypolactasia is associated with the transcriptional regulation of the LCT gene. The presence of the T-13910 A-22018 allele also showed significant elevation lactase activity. Galactose, the hydrolysing product of the milk sugar lactose, has been hypothesized to be poisonous to ovarian epithelial cells. Hence, consumption of dairy products and lactase persistence has been proposed to be a risk factor for ovarian carcinoma. To investigate whether lactase persistence is related to the risk of ovarian carcinoma the C/T-13910 genotype was determined in a cohort of 782 women with ovarian carcinoma 1331 individuals serving as controls. Lactase persistence did not associate significantly with the risk for ovarian carcinoma in the Finnish, in the Polish or in the Swedish populations. The findings do not support the hypothesis that lactase persistence increases the risk for ovarian carcinoma.

Nephrin and the Pathogenesis of Nephropathy - Emphasis on Type I Diabetes

End-stage renal disease is an increasingly common pathologic condition, with a current incidence of 87 per million inhabitants in Finland. It is the end point of various nephropathies, most common of which is the diabetic nephropathy. This thesis focuses on exploring the role of nephrin in the pathogenesis of diabetic nephropathy. Nephrin is a protein of the glomerular epithelial cell, or podocyte, and it appears to have a crucial function as a component of the filtration slit diaphragm in the kidney glomeruli. Mutations in the nephrin gene NPHS1 lead to massive proteinuria. Along with the originally described location in the podocyte, nephrin has now been found to be expressed in the brain, testis, placenta and pancreatic beta cells. In type 1 diabetes, the fundamental pathologic event is the autoimmune destruction of the beta cells. Autoantibodies against various beta cell antigens are generated during this process. Due to the location of nephrin in the beta cell, we hypothesized that patients with type 1 diabetes may present with nephrin autoantibodies. We also wanted to test whether such autoantibodies could be involved in the pathogenesis of diabetic nephropathy. The puromycin aminonucleoside nephrosis model in the rat, the streptozotocin model in the rat, and the non-obese diabetic mice were studied by immunochemical techniques, in situ -hybridization and the polymerase chain reaction -based methods to resolve the expression of nephrin mRNA and protein in experimental nephropathies. To test the effect of antiproteinuric therapies, streptozotocin-treated rats were also treated with aminoguanidine or perindopril. To detect nephrin antibodies we developed a radioimmunoprecipitation assay and analyzed follow-up material of 66 patients with type 1 diabetes. In the puromycin aminonucleoside nephrosis model, the nephrin expression level was uniformly decreased together with the appearance of proteinuria. In the streptozotocin-treated rats and in non-obese diabetic mice, the nephrin mRNA and protein expression levels were seen to increase in the early stages of nephropathy. However, as observed in the streptozotocin rats, in prolonged diabetic nephropathy the expression level decreased. We also found out that treatment with perindopril could not only prevent proteinuria but also a decrease in nephrin expression in streptozotocin-treated rats. Aminoguanidine did not have an effect on nephrin expression, although it could attenuate the proteinuria. Circulating antibodies to nephrin in patients with type 1 diabetes were found, although there was no correlation with the development of diabetic nephropathy. At diagnosis, 24% of the patients had these antibodies, while at 2, 5 and 10 years of disease duration the respective proportions were 23%, 14% and 18%. During the total follow-up of 16 to 19 years after diagnosis of diabetes, 14 patients had signs of nephropathy and 29% of them tested positive for nephrin autoantibodies in at least one sample. In conclusion, this thesis work could show changes of nephrin expression along with the development of proteinuria. The autoantibodies against nephrin are likely generated in the autoimmune process leading to type 1 diabetes. However, according to the present work it is unlikely that these autoantibodies are contributing significantly to the development of diabetic nephropathy.

The molecular basis of Marinesco-Sjögren syndrome

Marinesco-Sjögren syndrome (MSS) is a rare autosomal recessive neurodegenerative disorder characterized by cerebellar ataxia due to cerebellar cortical atrophy, infantile- or childhood-onset bilateral cataracts, progressive myopathy, and mild to severe mental retardation. Additional features include hypergonadotropic hypogonadism, various skeletal abnormalities, short stature, and strabismus. The neuroradiologic hallmarks are hypoplasia of both the vermis and cerebellar hemispheres. The histopathologic findings include severe cerebellar atrophy and loss of Purkinje and granule cells. The common pathologic findings in muscle biopsy are variation in muscle fiber size, atrophic fibers, fatty replacement, and rimmed vacuole formation. The presence of marked cerebellar atrophy with myopathy distinguishes MSS from another rare syndrome, the congenital cataracts, facial dysmorphism, and neuropathy syndrome (CCFDN). Previously, work by others had resulted in the identification of an MSS locus on chromosome 5q31. A subtype of MSS with myoglobinuria and neuropathy had been linked to the CCFDN locus on chromosome 18qter, at which mutations in the CTDP1 gene had been identified. We confirmed linkage to the previously identified locus on chromosome 5q31 in two Finnish families with eight affected individuals, reduced the critical region by fine-mapping, and identified SIL1 as a gene underlying MSS. We found a common homozygous founder mutation in all Finnish patients. The same mutation was also present in patient samples from Norway and Sweden. Altogether, we identified eight mutations in SIL1, including nonsense, frameshift, splice site alterations, and one missense mutation. SIL1 encodes a nucleotide exchange factor for the endoplasmic reticulum (ER) resident heat-shock protein 70 chaperone GRP78. GRP78 functions in protein synthesis and quality control of the newly synthesized polypeptides. It senses and responds to stressful cellular conditions. We showed that in mice, SIL1 and GRP78 show highly similar spatial and temporal tissue expression in developing and mature brain, eye, and muscle. Studying endogenous proteins in mouse primary hippocampal neurons, we found that SIL1 and GRP78 colocalize and that SIL1 localizes to the ER. We studied the subcellular localization of two mutant proteins, a missense mutant found in two patients and an artificial mutant lacking the ER retrieval signal, and found that both mutant proteins formed aggregates within the ER. Well in line with our findings and the clinical features of MSS, recent work by Zhao et al. showed that a truncation of SIL1 causes ataxia and cerebellar Purkinje cell loss in the naturally occurring woozy mutant mouse. Prior to Purkinje cell degeneration, the unfolded protein response is initiated and abnormal protein accumulations are present. MSS thus joins the group of protein misfolding and accumulation diseases. These findings highlight the importance of SIL1 and the role of the ER in neuronal function and survival. The results presented in this thesis provide tools for the molecular genetic diagnostics of MSS and give a basis for future studies on the molecular pathogenesis of MSS. Understanding the mechanisms behind this pleiotropic syndrome may provide insights into more common forms of ataxia, myopathy, and neurodegeneration.

Characterization of the TRIM37 gene and mutations underlying mulibrey nanism

Mulibrey nanism is a hereditary developmental disorder, characterized by prenatal onset growth failure without postnatal catch-up growth, distinctive craniofacial features, progressive cardiopathy and failure of sexual maturation. In addition, the patients develop insulin resistance syndrome and type 2 diabetes and they have an increased risk of developing tumors. The TRIM37 gene that underlies mulibrey nanism encodes for a member of the tripartite motif (TRIM) protein family. The physiological function of TRIM37 and the pathogenetic mechanisms leading from TRIM37 dysfunction to the mulibrey nanism phenotype are unknown. However, TRIM37 localizes at least partially to peroxisomes, and possesses ubiquitin E3-ligase activity. Thus, it may mediate ubiquitin dependent protein degradation, suggesting that accumulation of yet unknown substrate proteins may underlie the disease pathogenesis. In this study, the TRIM37 gene was characterized in detail. A transcription initiation window, with several separate transcription start sites, was identified and the putative promoter region immediately upstream from the transcription initiation window was shown to possess basal promoter activity. Further, several alternative splice variants of the gene were identified, including a highly expressed testis specific variant, encoding for an identical protein product with the main transcript. Expression of TRIM37 mRNA was detected in several different tissues, with highest expression seen in testis and in brain, when the expression patterns of the two major transcripts in different human tissues were studied by quantitative real-time PCR. Several mulibrey nanism patients were studied and thirteen novel mutations in TRIM37 were found, including three mutations (p.Gly322Val, p.Cys109Ser, p.Glu271_Ser287), that are likely to express mutant TRIM37 proteins. These mutations were further shown to alter the subcellular localization of the mutant proteins. Most of the mulibrey nanism associated mutations however, lead to premature termination codons and degradation of mRNA. All the TRIM37 mutations identified to date predict loss-of-function alleles, and thus no phenotype-genotype correlation is seen among the patients. In order to understand the pathogenetic mechanisms underlying mulibrey nanism, an animal model for the disorder is needed. For the development of a Trim37 knock-out mouse, the mouse Trim37 gene was characterized. Alternative splice variants, were identified, including a testis specific variant predicting a longer protein product. Further, a strictly tissue and cell-specific pattern of Trim37 expression was observed in developing and adult mouse tissues, when studied by immunohistochemical methods. This distribution of Trim37 expression in mouse tissues is in agreement with the clinical findings in human mulibrey nanism patients. This thesis work gives new tools for the diagnostics of mulibrey nanism as well as for studying the molecular pathogenesis behind this interesting disorder.

Small nerve fiber quantification in the diagnosis of diabetic sensorimotor polyneuropathy: Comparing corneal confocal microscopy with intraepidermal nerve fiber density

OBJECTIVE Quantitative assessment of small fiber damage is key to the early diagnosis and assessment of progression or regression of diabetic sensorimotor polyneuropathy (DSPN). Intraepidermal nerve fiber density (IENFD) is the current gold standard, but corneal confocal microscopy (CCM), an in vivo ophthalmic imaging modality, has the potential to be a noninvasive and objective image biomarker for identifying small fiber damage. The purpose of this study was to determine the diagnostic performance of CCM and IENFD by using the current guidelines as the reference standard. RESEARCH DESIGN AND METHODS Eighty-nine subjects (26 control subjects and 63 patients with type 1 diabetes), with and without DSPN, underwent a detailed assessment of neuropathy, including CCM and skin biopsy. RESULTS Manual and automated corneal nerve fiber density (CNFD) (P < 0.0001), branch density (CNBD) (P < 0.0001) and length (CNFL) (P < 0.0001), and IENFD (P < 0.001) were significantly reduced in patients with diabetes with DSPN compared with control subjects. The area under the receiver operating characteristic curve for identifying DSPN was 0.82 for manual CNFD, 0.80 for automated CNFD, and 0.66 for IENFD, which did not differ significantly (P = 0.14). CONCLUSIONS This study shows comparable diagnostic efficiency between CCM and IENFD, providing further support for the clinical utility of CCM as a surrogate end point for DSPN.

Sperm protamine deficiency correlates with sperm DNA damage in Bos indicus bulls

The primary purpose of spermatozoa is to deliver the paternal DNA to the oocyte at fertilization. During the complex events of fertilization, if the spermatozoon penetrating the oocyte contains compromised or damaged sperm chromatin, the subsequent progression of embryogenesis and foetal development may be affected. Variation in sperm DNA damage and protamine content in ejaculated spermatozoa was reported in the cattle, with potential consequences to bull fertility. Protamines are sperm-specific nuclear proteins that are essential to packaging of the condensed paternal genome in spermatozoa. Sperm DNA damage is thought to be repaired during the process of protamination. This study investigates the potential correlation between sperm protamine content, sperm DNA damage and the subsequent relationships between sperm chromatin and commonly measured reproductive phenotypes. Bos indicus sperm samples (n = 133) were assessed by two flow cytometric methods: the sperm chromatin structure assay (SCSA) and an optimized sperm protamine deficiency assay (SPDA). To verify the SPDA assay for bovine sperm protamine content, samples collected from testis, caput and cauda epididymidis were analyzed. As expected, mature spermatozoa in the cauda epididymidis had higher protamine content when compared with sperm samples from testis and caput epididymidis (p < 0.01). The DNA fragmentation index (DFI), determined by SCSA, was positively correlated (r = 0.33 ± 0.08, p < 0.05) with the percentage of spermatozoa that showed low protamine content using SPDA. Also, DFI was negatively correlated (r = -0.21 ± 0.09, p < 0.05) with the percentage of spermatozoa with high protamine content. Larger scrotal circumference contributes to higher sperm protamine content and lower content of sperm DNA damage (p < 0.05). In conclusion, sperm protamine content and sperm DNA damage are closely associated. Protamine deficiency is likely to be one of the contributing factors to DNA instability and damage, which can affect bull fertility. © 2014 American Society of Andrology and European Academy of Andrology.

Psychological consequences of false-positive screening mammograms in the UK

- Objectives To identify the psychological effects of false-positive screening mammograms in the UK. - Methods Systematic review of all controlled studies and qualitative studies of women with a false-positive screening mammogram. The control group participants had normal mammograms. All psychological outcomes including returning for routine screening were permitted. All studies had a narrative synthesis. - Results The searches returned seven includable studies (7/4423). Heterogeneity was such that meta-analysis was not possible. Studies using disease-specific measures found that, compared to normal results, there could be enduring psychological distress that lasted up to 3 years; the level of distress was related to the degree of invasiveness of the assessment. At 3 years the relative risks were, further mammography, 1.28 (95% CI 0.82 to 2.00), fine needle aspiration 1.80 (95% CI 1.17 to 2.77), biopsy 2.07 (95% CI 1.22 to 3.52) and early recall 1.82 (95% CI 1.22 to 2.72). Studies that used generic measures of anxiety and depression found no such impact up to 3 months after screening. Evidence suggests that women with false-positive mammograms have an increased likelihood of failing to reattend for routine screening, relative risk 0.97 (95% CI 0.96 to 0.98) compared with women with normal mammograms. - Conclusions Having a false-positive screening mammogram can cause breast cancer-specific distress for up to 3 years. The degree of distress is related to the invasiveness of the assessment. Women with false-positive mammograms are less likely to return for routine assessment than those with normal ones.

A Need for FSH in Maintaining Fertility of Adult Male Subhuman Primates

Adult fertile male bonnet monkeys (Macaca radiata) were continuously deprived of endogenous follicle stimulating hormone (FSH) support for 240 days by injecting them with 1 ml of characterized monkey antiserum to oFSH every 48 hr; control monkeys received during the same period normal monkey serum instead. Testicular function was assessed at periodic intervals by (a) carrying out differential counting of sperm in the ejaculate obtained and (b) determining the hyaluronidase activity as well as in vitro 3H thymidine incorporation into DNA of testicular tissue removed at biopsy. Both the quality (viability and motility) of the sperms voided and the total sperm counts showed marked decreases as a function of time of immunization, the first significant reduction being noted by 100 days. FSH deprivation affected both the biochemical parameters used to test testicular functionality they being reduced at ∼200 days by 50%-60%. The fertility of these monkeys was evaluated at periodic times after 90 days of treatment by means of mating studies. FSH deprivation had rendered the monkeys incapable of impregnating any of the females used. Testosterone and luteinizing hormone (LH) levels remained unchanged following FSH antiserum injection. With cessation of antiserum treatment testicular function and fertility were completely restored to normalcy, indicating that the observed effect was specifically due to FSH deprivation. This study thus provides conclusive evidence for the involvement of FSH in maintenance of testicular function and fertility in the adult male primate.

Regulation of Testosterone Rhythmicity by Gonadotropins in Bonnet Monkeys (Macaca radiata)

In an attempt to study the factor(s) that regulates production of nychthemeral testosterone surges in adult male bonnet monkeys (Macaca radiata), serum levels of testosterone, LH, FSH, and prolactin were monitored during a 24 h period. Only prolactin showed a significant increment in its levels coincident with that of the testosterone surge. The relationship between LH and testosterone production was studied by 1) observing the responsiveness of testes, in terms of testosterone production, to one or two injections of oLH (1 mg/injection) given 12 h apart at 0900 and 2100 h; and 2) monitoring the effect on testicular testosterone production of LH antiserum injection given at 1000, 1700, and 2100 h. That each LH injection brought about an increment in testosterone level of equal magnitude suggests that the difference in responsiveness of the testes to unchanging levels of LH at morning and night hours is not due to any alteration in substrate availability at the two time intervals. The LH antiserum experiments indicate that irrespective of the time of its administration the nocturnal surge of testosterone, which normally occurs at 2200 h, is blocked. While the antiserum prevents a rise in testosterone level, it appears not to influence basal testosterone production. The results further show that even at 2100 h, when surge testosterone production is already initiated, the testis is still highly sensitive to lack of LH, antiserum injection bringing about within 2 h a significant reduction in testosterone levels (by 69% in experimentals vs 11% in controls).