Female-specific Protein Research Articles

Novel female-specific splice form of dsx in the silkworm, Bombyx mori

The Bombyx mori doublesex (Bmdsx), a homologue of doublesex of Drosophila, is the bottom most gene of the sex determination cascade. Bmdsx plays a very crucial role in somatic sexual development. Its pre-mRNA sex-specifically splices to generate two splice variants; one encodes female-specific and the other encodes male-specific polypeptides which differ only at their C-termini. The open reading frame of Bmdsx consists of 5 exons, of which exons 3 and 4 are female-specific and are skipped in males. In the present study, we have identified a third splice form of the Bmdsx which is specific only to females and differs from the previously reported Bmdsxf isoform by the presence of 15 bp sequence. This new female splice form is generated as a result of alternative 5' splice site selection in the third exon adding additional 15 bp sequence in exon 3 which results in alteration of the reading frame leading to incorporation of an early stop codon. Thus the protein encoded by this splice form is 20 aa shorter than the known BmDsxF. Initial results obtained from the study of dsx homologues in Saturniid silkmoths suggest that both the female-specific Dsx proteins are essential for female sexual differentiation. It remains to be seen whether female-specific multiple splice forms of dsx are characteristic feature of only silkmoths or widespread among lepidopterans. The findings that sex determination mechanism is unique in lepidopterans offer an opportunity to develop genetic sexing methods in beneficial as well as economically destructive lepidopteran pests.

A rapid latex agglutination test for gender identification in the Atlantic bluefin tuna, Thunnus thynnus (Linnaeus)

A rapid, one-step agglutination assay has been developed, based on latex particles sensitized with antibodies against vitellogenin (Vtg), aimed at Atlantic bluefin tuna, Thunnus thynnus (Linnaeus) (ABFT), gender identification. The egg-yolk precursor protein Vtg was used as a gender marker for the assay as it is a female-specific protein synthesized during reproductive maturation. The presence of Vtg in the plasma was revealed in 60-120 s through an agglutination reaction by mixing small volumes of ABFT plasma and an anti-Vtg antibody-latex suspension on a microscope slide. The effectiveness of the present test was restricted to the months of May and June, concomitant with high circulating Vtg levels. Because of its rapidity and ease of performance in the field, the present gender identification assay could be useful for broodstock management in the aquaculture industry as well as in tagging studies on wild populations.

Two female-specific DSX proteins are encoded by the sex-specific transcripts of dsx, and are required for female sexual differentiation in two wild silkmoth species, Antheraea assama and Antheraea mylitta (Lepidoptera, Saturniidae)

doublesex ( dsx) is the bottom most gene of the sex-determination cascade of Drosophila melanogaster. The pre-mRNA of dsx splices to produce male- and female-specific transcripts which code for the male- and female-specific proteins, respectively. dsx homologues have been characterized from different (many in Diptera, two in Hypmenoptera and only one in Lepidoptera) insect species. Sex-specific splice forms of dsx pre-mRNA in all these species code for one male- and one female-specific DSX proteins, which regulate the downstream target genes responsible for sex-specific characters. In the present study we have cloned and characterized the dsx homologues from two saturniid silkmoths, Antheraea assama and Antheraea mylitta. The divergence time between Saturniidae and Bombycidae to which the domesticated silkworm, Bombyx mori belongs is estimated to be around 160.9 MY. Interestingly, the dsx pre-mRNA of these wild silkmoths sex-specifically splices to generate multiple splice variants. On the basis of their open reading frame (ORF) and conceptual translation, two female-specific (DSX F1 and DSX F2) and one male-specific (DSX M) proteins could be inferred, in both the moths. Presence or absence of a 15 bp stretch within the ORF of the two groups of female-specific transcripts resulted in the production of two distinct female-specific DSX proteins. The sex-specific DSX proteins have common amino-terminal sequence but sex-specific carboxy termini. The two female-specific DSX proteins (DSX F1 and DSX F2) share common DNA binding domain (DM domain) and oligomerization domain (OD domain) and differ only at their extreme C-termini by 21aa. Functional analysis of dsx transcripts in A. assama by dsRNA mediated knock-down resulted in complete abolition of expression of vitellogenin and hexamerin genes, the direct targets of the DSX proteins, irregular differentiation of gonads, and drastic reduction in fecundity and hatchability. Together, these results suggest the involvement of both the female-specific DSX proteins in the process of female sexual differentiation. Further, conservation of the 4th exon sequence, especially the PESS sequence responsible for the sex-specific splicing of Bmdsx in the female-specific transcripts of Aadsx and Amydsx, indicated the existence of a common mechanism of sex-specific splicing of dsx homologues in silkmoths. To our knowledge this is the first report of existence of multiple splice forms of dsx pre-mRNA encoding two female-specific DSX proteins.

Sex on the Fly

Sex on the Fly

The proteomic analysis of human neonatal umbilical cord serum by mass spectrometry

To investigate the proteome composition and function of human neonatal arterial umbilical cord. Serum proteomic analyses were performed on samples from both males and females by using a combination of techniques: (1) removal of six high-abundance proteins, (2) tryptic digestion of low-abundance proteins, (3) separation of peptide mixtures by reverse-phase high-performance liquid chromatography (RP-HPLC), and (4) peptide identification using electrospray ionization tandem mass spectrometry (ESI-MS/MS). A total of 837 non-redundant proteins were identified, with 213 male-specific and 239 female-specific proteins. Among them, 319 proteins were identified by at least 2 distinct peptides. The subcellular localization, function, and pathway involvement for each of the identified proteins were analyzed. A comparison of this neonatal proteome to that of adult serum proteome revealed novel biomarkers, such as alpha-fetoprotein and periostin that were specific to newborn infants. These data will contribute to a better understanding of the composition of umbilical cord serum and aid the discovery of novel biomarkers for the prenatal diagnosis of fetal abnormalities.

BmHrp28 is a RNA-binding protein that binds to the female-specific exon 4 ofBombyx mori dsxpre-mRNA

The Bombyx mori sex determination gene Bmdsx is alternatively spliced in the male and female to produce the male- and female-specific proteins. In an effort to better understand the mechanism of the alternative splicing regulation of Bmdsx, we conducted a gel-shift assay followed by LC-MS/MS analysis to identify the putative proteins bound to the cis-element CE1+6 in the exon 4 of Bmdsx. A protein named as BmHrp28 which is homologous to the Drosophila Hrp48, a member of the hnRNPA/B family, was identified and expressed in Escherichia coli for testing RNA-protein binding in vitro. All of the results showed that BmHrp28 specifically bound to the CE1+6 RNA probe. BmHrp28 has two RNA recognition motifs at the N-terminal and a glycine-rich motif at the C-terminal. It might be one of the factors involved in the male-specific splicing of Bmdsx.

Sex-specific concentrations of plasma proteins in rats determined by sex hormones in perinatal life.

The present investigation examined the relationship between concentrations of plasma proteins in oestrogen-primed adult rats and sex hormone concentrations in neonatal life. alpha 2-acute phase globulin (alpha 2-AP) and female specific protein (FP) were found to be higher in adult females than in adult males. Androgen administration in females on the first day of life declined permanently these protein concentrations, while neonatal castration in males on the first or third day of life resulted in a permanent increase in FP concentration. Both the alpha 2-AP and FP response in adulthood to hormonal stimulation is dependent on the androgen level during a critical neonatal organization period.

RNA structure: new messages in translation, replication and disease

The ‘Current Trends in Biomedicine’ workshop on the role of RNA structures in the translation of viral and cellular RNAs took place between 27 and 29 October 2008, in Baeza, Spain, and was organized by E. Martinez‐Salas, G. Belsham & J. Gomez. ![][1] See Glossary for abbreviations used in this article Since the elucidation of the genetic code approximately 40 years ago, the role of RNA in crucial biological processes has gained increasing attention, and is now one of the fastest developing areas in molecular biology and biochemistry. The advances made in structural studies of transfer RNA (tRNA), ribozymes, riboswitches, the ribosome and viral IRES elements in particular have shown the importance of secondary and tertiary structures in RNA biology, and have significantly changed our understanding of the translational control of gene expression (Fig 1). The World Heritage town of Baeza in Andalucia, Spain, hosted the ‘Current Trends in Biomedicine’ workshop on the role of RNA structures in the translation of viral and cellular RNAs. About 45 participants met in the beautiful buildings of the Sede Antonio Machado at the Universidad Internacional de Andalucia. Figure 1. Schematic illustration of discreet RNA regulatory elements. The translation of an mRNA is regulated by diverse mechanisms that involve both structural and non‐structural RNA elements, as well as interactions with RNA‐binding proteins. IRESes are found in the 5′ UTR and promote cap‐independent translation. Pseudoknots can be located in the 5′ UTR, the 3′ UTR, or the coding region, and their localization influences their effect on translation; for example, initiation, frameshifting and termination. 3′ UTR structural elements such as CREs, CITEs and SLs often function through long‐range RNA interactions. The miRNA target sites are located in the 3′ UTR. The canonical mRNA features of the cap (m7G) and poly(A) are targets of several regulatory interactions. The … [1]: /embed/graphic-1.gif

Characterization of chemoreceptive protein binding to an oviposition stimulant using a fluorescent micro-binding assay in a butterfly

A native female-specific chemoreceptive protein of a swallowtail butterfly [oviposition stimulant binding protein (OSBP)] was shown to specifically bind to aristolochic acid, a main stimulant for oviposition from its host plant. Oviposition stimulants are recognized by chemoreceptive organs of insects. OSBP isolated previously from the chemoreceptive organs was assumed to bind to an oviposition stimulant. Using a highly sensitive fluorescent micro-binding assay, we clarified OSBP bound to aristolochic acid. Three-dimensional molecular modeling revealed the structure of the OSBP-aristolochic acid complex. This is the first report of a native chemoreceptive protein binding to an oviposition stimulant as a ligand in insects.

Effects of starvation on the vitellogenesis, ovarian development and fecundity in the ectoparasitoid, Nasonia vitripennis (Hymenoptera: Pteromalidae)

AbstractA female‐specific protein, vitellogenin (Vg), and its corresponding egg vitellin (Vt) are identified in the ectoparasitic wasp Nasonia vitripennis. Both native Vt and Vg have a molecular mass of about 350 kDa, which is composed of two subunits of approximately 190 kDa and 165 kDa under reducing and denaturing conditions (sodium dodecyl sulfate—polyacrylamide gel electrophoresis). An indirect sandwich enzyme‐linked immunosorbent assay developed with both monoclonal and polyclonal antibodies against N. vitripennis Vt. Vg was first detected in the hemolymph on the 10th day after parasitism, and was first observed in oocytes on the 12th day. In adults deprived of food, the highest hemolymph Vg level occurred at the time of adult eclosion and the highest level of Vt in ovaries was found at 30 h after eclosion. In contrast, feeding adults with 20% sucrose resulted in the reduction of Vt uptake by ovaries and the extension of life span, but had little effect on Vg production. Deprived of hosts, starvation of female wasps had no significant effects on ovariole growth and oocyte maturation before the wasps died. However, starvation of female wasps supplied with hosts accelerated the wasps laying progeny into hosts, but resulted in a decrease of total progeny production by comparison with wasps fed with 20% sucrose.

Expression and localization of storage protein 1 (SP1) in differentiated fat body tissues of red hairy caterpillar,Amsacta albistrigaWalker

The accumulation and utilization of storage proteins are prominent events linked to the metamorphosis of holometabolous insects. The female-specific storage protein 1 (SP1) is the major storage protein found in the hemolymph and fat body of female larvae of the groundnut pest, Amsacta albistriga. Here we show SP1 expression and localization in differentiated fat body tissues using biochemical and immunohistochemistry scrutiny. Comparison of A. albistriga SP1 with that of other species with respect to amino acid composition and N-terminal sequences show that SP1 is a methonine-rich protein and its identity was confirmed by means of immunoblot analysis. Northern blot studies revealed that the SP1 gene demonstrates stage- and tissue-specific expression in the peripheral fat body cells during the mid-larval period of fifth instar of A. albistriga. During the larval pupal transformation, SP1 are sequestered mainly by the perivisceral fat body tissues, until they serve the purpose of supplying amino acids for the production of egg yolk proteins. Further, electron microscopic studies using immunogold tracer techniques confirmed the localization of crystalline SP1 reserves, stored in the perivisceral fat body tissues. Hence, the peripheral fat body is responsible for biosynthesis of storage proteins, whereas the perivisceral fat body is a specialized storage organ.

Structure, Function, and Evolution of Biogenic Amine-binding Proteins in Soft Ticks

Two highly abundant lipocalins, monomine and monotonin, have been isolated from the salivary gland of the soft tick Argas monolakensis and shown to bind histamine and 5-hydroxytryptamine (5-HT), respectively. The crystal structures of monomine and a paralog of monotonin were determined in the presence of ligands to compare the determinants of ligand binding. Both the structures and binding measurements indicate that the proteins have a single binding site rather than the two sites previously described for the female-specific histamine-binding protein (FS-HBP), the histamine-binding lipocalin of the tick Rhipicephalus appendiculatus. The binding sites of monomine and monotonin are similar to the lower, low affinity site of FS-HBP. The interaction of the protein with the aliphatic amine group of the ligand is very similar for the all of the proteins, whereas specificity is determined by interactions with the aromatic portion of the ligand. Interestingly, protein interaction with the imidazole ring of histamine differs significantly between the low affinity binding site of FS-HBP and monomine, suggesting that histamine binding has evolved independently in the two lineages. From the conserved features of these proteins, a tick lipocalin biogenic amine-binding motif could be derived that was used to predict biogenic amine-binding function in other tick lipocalins. Heterologous expression of genes from salivary gland libraries led to the discovery of biogenic amine-binding proteins in soft (Ornithodoros) and hard (Ixodes) tick genera. The data generated were used to reconstruct the most probable evolutionary pathway for the evolution of biogenic amine-binding in tick lipocalins.

Effects of 4‐nonylphenol on the endocrine system of the shore crab, Carcinus maenas

There is a considerable body of evidence to suggest that many anthropogenic chemicals, most notably xeno-estrogens, are able to disrupt the endocrine system of vertebrates. There have been few comparable studies on the effects of exposure to these chemicals that may serve as biomarkers of endocrine disruption in aquatic invertebrate species. In addition, the evidence available is complex, conflicting, and far from conclusive. The present study aimed to investigate the impact of the xeno-estrogen 4-nonylphenol (4-NP, nominal concentrations 10-100 microg L(-1)) on the regulation and functioning of the endocrine system of the shore crab Carcinus maenas. It also set out to establish whether 4-NP are causing the effects (i.e., changes of exoskeletons including secondary sexual characteristics, pheromonally mediated behavior and ecdysone levels, and the presence of vt in the male hepatopancreas) found recently in wild shore crabs (Lye et al.,2005). The study utilizes morphological (e.g., gonadosomatic and hepatosomatic indices) and hormonal (ecdysteroid moulting hormone levels and the induction of female specific proteins, vitellins) biomarkers using radioimmunoassay and an indirect enzyme linked immunosorbent assay applied to the soluble protein fraction of adult male hepatopancreatic homogenates. Exposure of C. maenas to an effective concentration as low as 1.5 microg L(-1) 4-NP resulted in a reduced testis weight, increased liver weight, and altered levels of ecdysone equivalents compared to controls. Induction of vitellin-like proteins was absent in all samples tested. The ecological implications and the possible mechanisms for the action of 4-NP on the response of the shore crab to xeno-estrogen exposure are discussed.

Lack of xenoestrogen-induced vitellogenin in male olive ridley sea turtles (Lepidochelys olivacea) from the Pacific coast of Costa Rica

Olive ridley sea turtles (Lepidochelys olivacea) distribute widely throughout Eastern Pacific (EP) waters. During reproduction these animals aggregate near coastal waters where they may be exposed to industrial or agrochemical environmental pollutants that may disrupt the endocrine system of these reptiles. One group of compounds of concern is the persistent organic pollutants (POPs), some of which have been detected in Costa Rican waters. Some of these POPs, such as polychlorinated biphenyls and DDT and its metabolites, exhibit estrogenic activity in reptiles. It is unknown whether olive ridleys are exposed to endocrine disruptive doses of these POPs throughout their life in EP waters. Consequently, this study was conducted to determine whether there is evidence of exposure of male olive ridleys to estrogenic compounds. During the study a total of 35 males were hand-captured while mating off the coast of Ostional, Costa Rica, a major nesting beach for this species. The captured turtles were brought aboard a small boat, and blood samples taken with a hypodermic syringe. Turtles were carefully released in the water after the blood samples were taken. In the laboratory, blood collected from these males was analyzed by Western blot using an antibody developed against red-eared slider turtle vitellogenin. Vitellogenin is a female-specific protein that is induced by estrogen during gonadal recrudescence. Estrogenic compounds have been shown to induce this protein in reptilian species. Results of this study indicate the lack of evidence of vitellogenin in the blood of olive ridley males as shown by Western blot analysis. This study demonstrates the feasibility of using the slider’s vitellogenin antibody to detect the presence of the protein in the olive ridley sea turtle, L. olivacea. Rev. Biol. Trop. 56 (Suppl. 4): 49-57. Epub 2009 June 30.

Male or Female? The Answer Depends on When You Ask

Male or Female? The Answer Depends on When You Ask

Purification of multiple vitellogenins in grey mullet (Mugil cephalus)

Three female specific serum proteins were detected immunologically in the sera of grey mullet (Mugil cephalus) which were named vitellogenin A (VgA), VgB, and VgC, based upon their distinct antigenicity against specific antisera raised against three types of mullet lipovitellins (Lvs). These Vgs were subsequently purified from the serum of estradiol-treated mullet by combining several types of chromatography columns (anion exchanger, hydroxylapatite, immunoadsorbent column, and gel filtration). Purified native VgA, VgB, and VgC exhibited molecular masses of 570, 580, and 335 kDa, respectively. Following, SDS-PAGE, the estimated mass of polypeptide bands evident for VgA and VgB were ∼179 and ∼175 kDa, respectively; VgC appeared to be ∼132 kDa. The two larger Vgs (VgA and VgB) appeared to be phosphorylated, suggesting that these Vgs contain a highly phosphorylated, serine-rich phosvitin (Pv) domain. Furthermore, two discrete Vg-type specific antisera, anti-VgA and anti-VgB, were developed and each generated two precipitin lines against ovary extracts in immunoelectrophoresis, indicating that these Vgs contain additional antigenic yolk protein domains: Lv and β′-component. The small Vg (VgC) appeared to lack a Pv domain because of its low serine content (5.35%) and failure to show positive results in phospho-staining experiments. In conjunction with N-terminal amino acid sequencing analyses of the purified Vgs, our present results have conclusively identified the purified Vg products in grey mullet as typical A-type (VgA), B-type (VgB), and C-type (VgC) Vgs.

Drosophila melanogasterMale Somatic Cells Feminized Solely by TraF Can Collaborate With Female Germ Cells to Make Functional Eggs

Female differentiation of Drosophila germ cells is induced by cell-nonautonomous signals generated in the gonadal soma that work with germ-cell-autonomous signals determined by germ-cell X chromosome dose. Generation of the nonautonomous feminizing signals was known to involve female-specific protein encoded by the master sex-determination gene Sex-lethal (Sxl) acting on its switch-gene target transformer (tra) to produce Tra(F) protein. However, it was not known whether Sxl's action on tra alone would suffice to trigger a fully feminizing nonautonomous signal. We developed a constitutively feminizing tra transgene that allowed us to answer this question. In gynanders (XX//XO mosaics) feminized by this Tra(F) transgene, functionally Sxl- haplo-X (chromosomally male) somatic cells collaborated successfully with diplo-X (chromosomally female) germ cells to make functional eggs. The fertility of such gynanders shows not only that Tra(F) is sufficient to elicit a fully feminizing nonautonomous signal, but also that haplo-X somatic cells can execute all other somatic functions required for oogenesis, despite the fact that their genome is not expected to be dosage compensated for such diplo-X-specific functions. The unexpected observation that some Tra(F)-feminized gynanders failed to lay their eggs showed there to be diplo-X cells outside the gonad for which Tra(F)-feminized haplo-X cells cannot substitute.

Nuclear receptors link gender dimorphism of renal disease progression

Profound gender differences in the progression of chronic kidney disease (CKD) remain poorly understood. Differential expression of the genes for male- and female-specific proteins in the kidney has been proposed to account for this clinical phenomenon. Lu and colleagues provide evidence that the signaling pathways of the nuclear receptors ERalpha, AR and PPARalpha are associated with gender differences in CKD progression.

Pfs47, paralog of the male fertility factor Pfs48/45, is a female specific surface protein in Plasmodium falciparum

The genome of Plasmodium falciparum contains a small gene family that expresses proteins characterized by the presence of 6-cysteine domains. Most of these proteins are expressed on the surface of the parasite and some are known to play a role in cell–cell interactions. Two members of this family, Pfs48/45 and Pfs230, form a complex localized on the surface of gametes and are recognized as important targets for transmission-blocking vaccines. In this study we report the analysis of an additional member of this family, Pfs47 the closest paralog of Pfs48/45. We demonstrate that Pfs47 is expressed only in female gametocytes and is located on the surface of female gametes following emergence from red blood cells. In contrast to the critical function of P48/45 for male fertility, Pfs47 does not appear crucial for female fertility. Parasites lacking Pfs47 through targeted gene disruption, produce normal numbers of oocysts when included in the blood meal of the mosquito vector. In addition, three monoclonal antibodies against Pfs47 were unable to inhibit oocyst development when present in a blood meal containing wild type parasites. These results show redundancy in protein function for Pfs47 and reduce the support for candidacy of Pfs47 as a transmission-blocking vaccine target.

UNRaveling the regulation of dosage compensation

Translational inhibition of male-specific msl-2 messenger RNA by the female-specific protein SXL is crucial for X-chromosome dosage compensation in Drosophila melanogaster. Two recent studies identify an RNA-binding protein, UNR, as a novel corepressor that is recruited by SXL to the 3' UTR of msl-2 mRNA for translation inhibition in females.