Review disorders of sex development: The evolving role of genomics in diagnosis and gene discovery

Disorders of Sex Development (DSDs) are a major paediatric concern and are estimated to occur in around 1.7% of all live births (Fausto‐Sterling, Sexing the Body: Gender Politics and the Construction of Sexuality, Basic Books, New York, 2000). They are often caused by the breakdown in the complex genetic mechanisms that underlie gonadal development and differentiation. Having a genetic diagnosis can be important for patients with a DSD: it can increase acceptance of a disorder often surrounded by stigma, alter clinical management and it can assist in reproductive planning. While Massively Parallel Sequencing (MPS) is advancing the genetic diagnosis of rare Mendelian disorders, it is not yet clear which MPS assay is best suited for the clinical diagnosis of DSD patients and to what extent other established methods are still relevant. To complicate matters, DSDs represent a wide spectrum of disorders caused by an array of different genetic changes, many of which are yet unknown. Here we discuss the different genetic lesions that are known to contribute to different DSDs, and review the utility of a range of MPS approaches for diagnosing DSD patients. Birth Defects Research (Part C) 108:337–350, 2016. © 2016 Wiley Periodicals, Inc.     

Biochemical analysis of programmed cell death during premeiotic stages of spermatogenesis in vivo and in vitro

Control points of regulator action during spermatogenesis are not completely known. Using the shark testis model, which facilitates analysis of spermatogenesis stage-by-stage in vivo and in vitro, an early biochemical marker of programmed cell death (PCD) was detected. Nucleosomal oligomers were seen in DNA extracts of testis and isolated spermatocysts (clonal germ cell/ Sertoli cell units) at premeiotic (PrM), but not meiotic (M) or postmeiotic (PoM), stages. Cell nuclei isolated from M stages of development were susceptible to cleavage by micrococcal nuclease, suggesting that developmental control of factors other than a nuclease-insensitive chromatin structure may account for stage specificity. Cytological features of apoptosis were seen in germ cells, but not Sertoli cells, of a subset of isolated PrM spermatocysts and appeared to be all-or-none in affected clones. In culture, DNA fragmentation occurred on schedule with or without various additives, but the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) decreased accumulation of DNA breakdown products. Identification of the apoptotic form of PCD as a major, variable component of normal spermatogenesis and the use of PrM spermatocysts as an in vitro test system will allow further definition of mechanisms and developmental and physiological controls. © 1995 Wiley-Liss, Inc.     

Partial purification and characterization of dipeptidyl peptidase II (DPP II) from guinea pig testes

Depeptidyl peptidase (DPP II) was partially purified from guinea pig testes by (NH₄)₂SO₄ precipitation, Con A-Sepharose 4B chromatography, and Sephadex G-200 chromatography to a specific activity of 27.4 μmol Ala₃ hydrolyzed min^?1 mg^?1 protein. Chromatography on a calibrated G-200 column yielded a molecular weight of 135,000 daltons for the enzyme. Sodium dodecyl sulfate polyacrylamide electrophoresis showed an enrichment of a broad doublet at 64–66,000 daltons. The enzyme had optimal activity toward hydrolysis of L-alanyl-alanyl-alanine at pH 4.5 and showed sensitivity to cations of increasing size with Tris producing the most inhibition of those tested. The enzyme was moderately inhibited by serine proteinase inhibitors. Thin-layer chromatography revealed the dipeptidase nature of the enzyme's activity on tripeptides and dipeptidyl arylamides. A doublet of activity occurred when nitrocellulose electroblots of nondenaturing gel electrophoresis of the (NH₄)₂SO₄ fraction were reacted with the specific DPP II substrate, lysyl-alanyl-4-methoxy-2-napthylamide. Analytical isoelectric focusing of the G-200 fraction followed by fluorescent enzyme activity detection that used cellulose triacetate overlay membranes impregnated with the specific DPP II substrate, lysyl-alanyl-7-amino-4-trifluoromethylcou-marin, revealed multiple isoforms focusing at pI = 4.8–5.6. Two prominent bands focused at pI = 4.9 and pI = 5.1. The properties of guinea pig testicular DPP II are compared and contrasted with similar dipeptidyl peptidases from other sources.     

Primates: Their use in research on vasectomy

Studies based on experimental vasectomies clearly reveal marked species differences in response to vasectomy. In rats, vasectomy invariably results in granuloma formation at the surgical site. In rabbits, immune-complex orchitis develops. Allergic orchitis may also develop in guinea pigs, and the morphological lesions can be adoptively transferred. My co-workers and I have been able to study systematically, biochemically, hormonally, pathologically, and immunologically, primates vasectomized up to 14 years earlier and to compare them with age-matched controls. We have monitored antibody levels in vasectomized rhesus and cynomolgus macaques by sperm-agglutination, sperm-immobilization, and indirect immunofluorescence techniques. Antibodies develop in almost every monkey, in some as early as 10 days after vasectomy. About 50% retain such circulating antisperm antibodies. In men, antibody development is less rapid, and about half of vasectomized men reveal detectable levels. Testicular histopathological studies have revealed detectable levels. Testicular histopathological studies have revealed orchitis, aspermatogenesis, or both, resembling allergic orchitis in most of the vasectomized monkeys and in about one-fourth of the controls. Limited studies of human material reveal some testicular changes. Epididymitis and epididymal granuloma occur exclusively in the vasectomized animals. Use of immunofluorescence has revealed significantly more granular deposits of IgA, IgG, and/or C3 in the basal lamina of the ductus efferens and the caput epididymidis of the vasectomized monkeys. Similar studies on human material have not been done. Both cynomolgus and rhesus macaques have proved to be excellent models for research in atherosclerosis and so have been used to determine whether constant sperm antigen leakage causes immune-complex formation that might result in arteritis and atherosclerosis. Evaluation of the cardiovascular systems from such animals has revealed that vasectomized monkeys have more frequent, more extensive, and more severe arteriosclerosis than age-matched controls. Epidemiological studies are currently under way to determine whether such an effect also occurs in men after vasectomy.     

Testis mediated gene transfer: In vitro transfection in goat testis by electroporation

Testis mediated gene transfer (TMGT) is a potential tool for making transgenic mice having more than 90% success rate. However, this method needs further standardization before it can be adapted in other species including livestock. In order to standardize the TMGT in goat, buck testes (n = 20) collected from the slaughter house were injected with a vector driving green fluorescent protein (GFP) expression under a cytomegalovirus (CMV) promoter. Then, the testes were subjected to electroporation with predetermined voltage, pulse length, pulse interval and number of pulses. Seminiferous tubules were isolated from the electroporated testis and cultured in-vitro. The expression was checked at regular intervals. Green fluorescence was observed on different days in different samples. It suggests transient integration of the plasmid into the seminiferous tubules. This in-vitro transfection of seminiferous tubule using electroporation will provide valuable baseline information.     

Whole‐body heat exposure causes developmental stage‐specific apoptosis of male germ cells

Humans are occasionally exposed to extreme environmental heat for a prolonged period of time. Here, we investigated testicular responses to whole‐body heat exposure by placing mice in a warm chamber. Among the examined tissues, the testis was found to be most susceptible to heat stress. Heat stress induces direct responses within germ cells, such as eukaryotic initiation factor 2α phosphorylation and stress granule (SG) formation. Prolonged heat stress (42°C for 6 hr) also disturbed tissue organization, such as through blood‐testis barrier (BTB) leakage. Germ cell apoptosis was induced by heat stress for 6 hr in a cell type‐ and developmental stage‐specific manner. We previously showed that spermatocytes in the early tubular stages (I–VI) form SGs for protection against heat stress. In the mid‐tubular stages (VII–VIII), BTB leakage synergistically enhances the adverse effects of heat stress on pachytene spermatocyte apoptosis. In the late tubular stages (IX–XII), SGs are not formed and severe leakage of the BTB does not occur, resulting in mild apoptosis of late‐pachytene spermatocytes near meiosis. Our results revealed that multiple stress responses are involved in germ cell damage resulting from prolonged heat stress (42°C for 6 hr).     

Inhibition by gossypol of phospholipid-sensitive Ca2+-dependent protein kinase from pig testis

Gossypol, a polyphenolic binaphthalene-dialdehyde extracted from cotton plants which possesses male antifertility action in mammals, is a potent inhibitor of phospholipid-sensitive Ca²⁺-dependent protein kinase from pig testis. Gossypol inhibited Ca²⁺-dependent activity of the enzyme without affecting its basal activity. The IC₅₀ value (concentration causing 50% inhibition) was 31 μM when lysine-rich histone was used as substrate. Kinetic analysis indicated that the compound inhibited the enzyme non-competitively with respect to ATP (K_i = 31 μM) or lysine-rich histone (K_i = 30μM), and competitively with respect to phosphatidylserine (K_i = 2.1 μM). With Ca²⁺, irrespective of the presence or absence of 1,3-diolein, the compound lowered V_max and increased the apparent K_a for Ca²⁺. The compound also inhibited phosphorylation by the enzyme of high-mobility-group 1 protein (one of the endogenous substrate in the testis for the enzyme located in nucleosome), with an IC₅₀ value of 88 μM. These results suggested that a phospholipid-sensitive Ca²⁺-dependent protein phosphorylation system in the testis is involved in the regulation of spermatogenesis.     

The Dynamic Transcriptional Cell Atlas of Testis Development during Human Puberty

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Changing Male Reproductive Health: A Review of the Clinical Evidence?

Several lines of circumstantial evidence suggest that we may be seeing adverse changes in male reproductive health. A possible decline in semen quality has attracted most attention, but there are stronger indications of a rising incidence of testicular cancer, with increases observed in both Europe and the USA. There are striking geographic variations in both the incidence of testis cancer and in the observed rate of increase, and it is noteworthy that testis cancer is much more common in Denmark, where low sperm counts have been reported, than in Finland, where semen quality seems to be better. Another cause for concern is the rising incidence of congenital malformations of the male genital tract — cryptorchidism and hypospadias. In the UK, for example, rates of cryptorchidism have increased by as much as 65 to 77%. The data are harder to interpret on semen quality. In a meta-analysis, Carlsen et al. (1992) identified significant decreases over time in sperm concentration, corresponding to a fall of almost 50% between 1940 and 1990. Several groups have since examined secular trends in semen quality, with some reporting a downward trend and others no change. However, evidence has emerged of striking regional differences in semen quality, whether due to ethnic, genetic, environmental, or lifestyle factors remains to be determined.