Claude M. Nagamine

Effects of androgenic gland ablation on male primary and secondary sexual characteristics in the Malaysian prawn, Macrobrachium rosenbergii (de Man) (Decapoda, Palaemonidae), with first evidence of induced feminization in a nonhermaphroditic decapod

Abstract Male Macrobachium rosenbergii (de Man), categorized into three developmental stages according to possession of male gonopore complexes, appendixes masculina, and mature chelipeds, were subjected to bilateral androgenic gland ablation (andrectomy). Andrectomized males initially lacking appendixes masculina and mature chelipeds do not develop them; those initially possessing appendixes masculina and mature chelipeds do not lose them. Growth rate of the appendixes masculina, however, is reduced. Andrectomized males are unable to redifferentiate amputated or accidentally lost appendixes masculina and mature chelipeds. Instead, the appendages regenerate as immature forms. Andrectomized M. rosenbergii possess atrophied testes and vasa deferentia. Histological sections of the testes revealed a reduction in number of spermatogenic lobules. Spermatogonia and primary spermatocytes were commonly found; secondary spermatocytes and spermatids were found only occasionally. Andrectomy appears to inhibit but not prevent meiosis. Feminization occurred in five andrectomized males. Complete feminization, including initiation of oogenesis and development of oviducts and female gonopores, occurred in males andrectomized in the youngest developmental stage. Males andrectomized in later developmental stages were either partially feminized or not feminized. Reimplantation of androgenic glands reverses the effects of andrectomy. Remasculinization, as evidenced by differentiation of appendixes masculina, was noted after the first postimplantation molt. The testes of remasculinized males were normal in gross anatomy and spermatogenic ability. The data indicate a uniform function for the androgenic glands with respect to male primary and secondary sexual characteristics among the Amphipoda, Isopoda, and Decapoda. Past theories on androgenic gland function are modified in light of the present information.

An essential receptor for adeno-associated virus infection

Adeno-associated virus (AAV) vectors are currently the leading candidates for virus-based gene therapies because of their broad tissue tropism, non-pathogenic nature and low immunogenicity. They have been successfully used in clinical trials to treat hereditary diseases such as haemophilia B (ref. 2), and have been approved for treatment of lipoprotein lipase deficiency in Europe. Considerable efforts have been made to engineer AAV variants with novel and biomedically valuable cell tropisms to allow efficacious systemic administration, yet basic aspects of AAV cellular entry are still poorly understood. In particular, the protein receptor(s) required for AAV entry after cell attachment remains unknown. Here we use an unbiased genetic screen to identify proteins essential for AAV serotype 2 (AAV2) infection in a haploid human cell line. The most significantly enriched gene of the screen encodes a previously uncharacterized type I transmembrane protein, KIAA0319L (denoted hereafter as AAV receptor (AAVR)). We characterize AAVR as a protein capable of rapid endocytosis from the plasma membrane and trafficking to the trans-Golgi network. We show that AAVR directly binds to AAV2 particles, and that anti-AAVR antibodies efficiently block AAV2 infection. Moreover, genetic ablation of AAVR renders a wide range of mammalian cell types highly resistant to AAV2 infection. Notably, AAVR serves as a critical host factor for all tested AAV serotypes. The importance of AAVR for in vivo gene delivery is further highlighted by the robust resistance of Aavr−/− (also known as Au040320−/− and Kiaa0319l−/−) mice to AAV infection. Collectively, our data indicate that AAVR is a universal receptor involved in AAV infection.

Masculinization of female Macrobrachium rosenbergii (de Man) (Decapoda, Palaemonidae) by androgenic gland implantation

Abstract Sexually immature and mature female Macrobrachium rosenbergii (de Man) were implanted with vas deferens, testicular, or androgenic gland tissues. Females implanted with vas deferens or testicular tissue developed normally. In contrast, androgenic gland implantation masculinized 81% of female recipients. The first morphological evidence of masculinization was usually differentiation of appendixes masculina. Male gonopore complexes were developed by three sexually immature females and mature male chelipeds were developed by six large masculinized females. Females that were sexually immature at masculinization, i.e., those lacking brood chambers and reproductive setae, did not develop these structures. Females that were sexually mature at masculinization still evidenced brood chambers and reproductive setae at the termination of the experiment. All masculinized females retained their female gonopores. Internally, all but one masculinized female possessed bilaterally formed vasa deferentia. In females possessing male gonopore complexes, the vasa deferentia were completely formed. In the other masculinized females, the vasa deferentia were incompletely formed and existed as autonomous growths along the cephalothorax wall. Two masculinized females initiated spermatogenesis in their ovaries. Spermatogenic lobules were generally better developed in the anterior region of the ovaries. Oogenesis was still present but the oocytes never completed vitellogenesis. In remaining masculinized females, no evidence of spermatogenesis was found. Oogenesis was present but, as in females that had initiated spermatogenesis, the oocytes never completed vitellogenesis. The present results are comparable to past results of androgenic gland implantation experiments and support our hypothesis that the androgenic glands function similarly in the Amphipoda, Isopoda, and Decapoda.

Inhibition of cellular autophagy deranges dengue virion maturation.

ABSTRACT Autophagy is an important component of the innate immune response, directly destroying many intracellular pathogens. However, some pathogens, including several RNA viruses, subvert the autophagy pathway, or components of the pathway, to facilitate their replication. In the present study, the effect of inhibiting autophagy on the growth of dengue virus was tested using a novel inhibitor, spautin-1 (specific and potent autophagy inhibitor 1). Inhibition of autophagy by spautin-1 generated heat-sensitive, noninfectious dengue virus particles, revealing a large effect of components of the autophagy pathway on viral maturation. A smaller effect on viral RNA accumulation was also observed. Conversely, stimulation of autophagy resulted in increased viral titers and pathogenicity in the mouse. We conclude that the presence of functional autophagy components facilitates viral RNA replication and, more importantly, is required for infectious dengue virus production. Pharmacological inhibition of host processes is an attractive antiviral strategy to avoid selection of treatment-resistant variants, and inhibitors of autophagy may prove to be valuable therapeutics against dengue virus infection and pathogenesis.

The musculus-type Y chromosome of the laboratory mouse is of Asian origin

Mus musculus domesticus, M.m. bactrianus, M. m. musculus, M.m. castaneus, and M.m. molossinus wild mice were investigated for polymorphisms of the Y Chromosome (Chr) genes Zinc finger-Y (Zfy) and Sex-determining region-Y (Sry). Zfy divided the Y Chrs of these mice into domesticus- (domesticus) and musculus-types (musculus, castaneus, molossinus). M.m. bactrianus specimens had both Y Chrs, possibly owing to the introgression of a musculus-type Y into this population. Sry identified a subpopulation of musculus-type Y chromosomes. This subpopulation, designated the molossinus-type, was found in M.m. molossinus, a M. musculus subspecies specimen from northern China (Changchun), and laboratory mice. The cumulative data suggest that M.m. musculus of northern China and Korea are subpopulation distinct from M.m. musculus of Europe and central China and that this subpopulation invaded Japan, giving rise to M.m. molossinus. Furthermore, the data suggest that the musculus-type Y of the laboratory mouse originated from this subpopulation, corroborating early historical record reporting that Chinese and Japanese mice that were imported into Europe for the pet trade contributed to the genome of the laboratory mouse.

The testis-determining gene, SRY , exists in multiple copies in Old World rodents

SRY is a unique gene on the Y chromosome in most mammalian species including the laboratory mouse, Mus musculus, and the closely related European wild mouse species M. spicilegus, M. macedonicus, and M. spretus. In contrast, SRY is present in 2-6 copies in the more distantly related Asian mouse species M. caroli, M. cervicolor, and M. cookii and in 2-13 copies in the related murid species Pyromys saxicola, Coelomys pahari, Nannomys minutoides, Mastomys natalensis, and Rattus norvegicus. Copy numbers do not correlate with known phylogenetic relationships suggesting that SRY has undergone a rapid and complex evolution in these species. SRY was recently proposed as a molecular probe for phylogenetic inferences. The presence of multiple SRY genes in a wide range of murid species and genera, and at least one cricetid species, necessitates caution in the use of SRY for phylogenetic studies in the Rodentia unless it is ascertained that multiple SRY genes do not exist.

Hst-3: an X-linked hybrid sterility gene.

A gene, Hst-3, responsible for sterility in F1 males from crosses between Mus spretus and laboratory strains of mice such as C57BL/6, has been localized on the distal part of the X chromosome, using both DNA probes and biochemical markers on a panel of F1(C57BL/6 x SEG) x C57BL/6 backcross males. This gene may be a model for studying mammalian hybrid sterility.

Helicobacter hepaticus Infection Promotes Colon Tumorigenesis in the BALB/c-Rag2 / Apc Min/ Mouse

ABSTRACT Adenomatous polyposis coli (APC) mutations are linked to human and mouse colorectal cancers. The Apc multiple intestinal neoplasia (Min) mouse mutation causes adenomas to develop throughout the small and large intestines. The BALB-Min (C.B6-ApcMin/+) congenic strain was generated by backcrossing into BALB/c the ApcMin allele from C57BL/6J-ApcMin/+ mice. BALB-Min mice have a low tumor multiplicity (27.4 small intestine tumors/mouse) and a relatively long life span (>1 year) that makes them amenable to long-term studies. To investigate the interplay of the adaptive immune system and intestinal tumorigenesis, the immunodeficient compound mutant strain BALB-RagMin (C.Cg-Rag2−/−ApcMin/+) was generated. BALB-RagMin mice had a significant increase in tumors in the small, but not large, intestine relative to their BALB-Min counterparts (43.0 versus 24.0 tumors/mouse, respectively). The results suggest that the adaptive immune system plays a role in either the elimination or the equilibrium phase of cancer immunoediting in the small intestine in this model. We investigated the effect of the enterohepatic bacterial pathogen Helicobacter hepaticus on liver and intestine tumorigenesis in BALB-RagMin mice. H. hepaticus-infected BALB-RagMin mice developed moderate hepatitis, moderate typhlitis, and mild colitis. There were no differences in small intestine and cecal tumor multiplicity, regionality, or size relative to that in uninfected mice. However, H. hepaticus-infected BALB-RagMin mice had a significant increase in colon tumor incidence relative to uninfected BALB-RagMin mice (23.5% versus 1.7%, respectively). The data suggest that H. hepaticus, which is present in many research colonies, promotes colon tumorigenesis in the BALB-RagMin mouse and that it has the potential to confound colon tumorigenesis studies.

Development, Maturation, and Function of Some Sexually Dimorphic Structures of the Malaysian Prawn, Macrobrachium Rosenbergii (De Man) (Decapoda, Palaemonidae)

The development, maturation, and function of some sexually dimorphic characteristics of Macrobrachium rosenbergii (De Man) were studied. The first evidence of sexual dimorphism is the appearance of gonopores. The smallest male had a carapace length (CL) of 5.9 mm; the smallest female had a CL of 7.6 mm. The endopods of the first pair of pleopods are sexually dimorphic, with those of the male being broad and flat, while those of the female are narrow, roundish, and possess long, fine setae. Sexual dimorphism of the endopods, as evidenced by a difference in the relative lengths of the exopod and endopod, occurs at about 12 mm CL. In the males, the appendices masculinae begin to develop at about 10 mm CL. By 13 mm CL, they attain a length of 1.7 times the length of neighboring appendices internae. This size relationship is generally maintained in future growth. Male chelipeds begin to mature at 28 mm CL, although males as large as 38 mm CL may still possess immature chelipeds. Maturation is associated with an increase in growth of mainly the merus through dactylus articles. The larger male chelipeds function primarily in reproduction by allowing a male: 1) to fend off other males intruding into its territory, and 2) to protect females, with which it has mated, from aggressive conspecifics and predators. A difference in age of maturation of the male chelipeds was noted between the present study and one done by Cowles (1914) in the Philippines. The possibility that the latter population may be a subspecies is discussed. The brood chamber and reproductive setae of females begin to develop at 20 mm CL. This may occur several molts before their first spawning. Reproductive setae are divided into ovigerous and ovipositing setae, depending upon their function. Ovigerous setae are temporary structures formed only during preparturial molts. While some ovipositing setae are also temporary, others are permanent since they are retained on nonparturial molts. A decrease in their length, however, may occur.

Masculinization of female crayfish, Procambarus Clarki (Girard)

Twenty-three immature female crayfish (Procambarus clarki (Girard); carapace length = 8.2–17.9 mm), implanted with androgenic gland implants obtained from P. clarki males, were observed for 326 day...