Mucilage secretion regulated by sex pheromones in Closterium peracerosum-strigosum-littorale complex

When mating-type plus (mt+) and minus (mt-) cells of the Closterium peracerosum-strigosum-littorale complex were mixed in nitrogen-depleted mating medium, secretion of mucilage containing uronic acid from cells was markedly activated and the mucilage accumulated around the cells. Substances with the ability to stimulate mucilage secretion from mt+ and mt- cells were detected in media in which mt- and mt+ cells had been separately cultured, respectively. We designated the active substances secreted from mt+ and mt- cells mucilage secretion-stimulating pheromone (MS-SP)-plus and MS-SP-minus, respectively. Activity of MS-SP-plus and MS-SP-minus decreased to 20% level by incubation at 80 degrees C for 10 min. Light was indispensable for the secretion of mucilage. The secretion of MS-SP-plus and MS-SP-minus decreased with dark treatment. MS-SP-plus eluted at around 95 k from a gel filtration column, and reacted with antibodies against two subunits of protoplast-release-inducing protein (PR-IP), which induces protoplast release from mt- cells. MS-SP-minus eluted at around 20 k from a gel filtration column, and reacted with an antibody against the PR-IP inducer, which induces the secretion of PR-IP from mt+ cells. In addition, purified PR-IP and PR-IP inducer stimulated mucilage secretion from mt- and mt+ cells, respectively. These results strongly suggested that MS-SP-plus and MS-SP-minus were the same molecules as the PR-IP and the PR-IP inducer, respectively.     

Production and secretion of a biologically active Closterium sex pheromone by Saccharomyces cerevisiae

The cDNA fragment coding for the Closterium sex pheromone (protoplast-release-inducing protein inducer, PR-IP inducer) was inserted into a Saccharomyces cerevisiae–Escherichia coli shuttle vector, under the control of the glucose-repressible alcohol dehydrogenase (ADH2) promoter of yeast. The yeast cells transformed by this construct produced and secreted recombinant PR-IP inducer into the surrounding culture medium. The release of PR-IP from mt⁺ cells was induced by the recombinant pheromone, whereas amino- and carboxy-terminal truncated forms did not show any biological activity. High levels of asparagine-linked glycosylation in the recombinant pheromone were also confirmed after the treatment by deglycosylation enzymes.     

SEX PHEROMONES THAT INDUCE SEXUAL CELL DIVISION IN THE CLOSTERIUM PERACEROSUM–STRIGOSUM–LITTORALE COMPLEX (CHAROPHYTA)1

Sexual cell division (SCD) that produces two gametangial cells from one vegetative mother cell is the first step observed morphologically in the sexual reproduction in the Closterium peracerosum–strigosum– littorale complex. SCD‐inducing activities specific for each mating‐type cells were detected in the medium in which both mating type cells has been cocultured. Mating‐type minus (mt ^? ) cells released SCD‐inducing substance specific for mating‐type plus (mt ⁺ ) cells and were designated as SCD‐ inducing pheromone (IP)‐minus, whereas mt ^? specific substances released from mt ⁺ cells were designated as SCD‐IP‐plus. Culture medium was subjected to gel filtration, and then SCD‐IP‐plus and SCD‐IP‐minus chemical were found to have the molecular masses of 90–100 kDa and 10–20 kDa, respectively. It was evident that light was imperative for this type of signaling. Gametangial cells of both mating types were obtained from vegetative cells by treatment with SCD‐IPs. Gametangial mt ⁺ cells showed high competency for conjugation with vegetative mt ^? cells, whereas gametangial mt ^? cells showed low competency for conjugation with vegetative mt ⁺ cells. These results indicate that SCD in both mating type cells is induced by high molecular weight sex pheromones and that the roles of gametangial cells in the process of conjugation differ by sex.     

Purification and characterization of a pheromone that induces sexual cell division in the unicellular green alga Closterium ehrenbergii

Closterium ehrenbergii is a unicellular charophycean alga consisting of two sexes: mating type plus (mt⁺) and minus (mt^–). The sexual reproductive process consists of five steps: formation of sexual pairs, cell division of each member of a pair, formation of conjugation papillae, release of protoplasts from gametangial cells, and fusion of protoplasts to form a zygote. The second step, called sexual cell division (SCD), produces two gametangial cells from one vegetative mother cell. The SCD of mt⁺ cell is mediated by a diffusible sex pheromone, named SCD-inducing pheromone (SCD-IP). This pheromone is released from mt^– cells in the light, and the presence of mt⁺ cells stimulates its secretion from mt^– cells. SCD-IP was purified by sequential column-chromatographic fractionation from culture medium in which both mating type cells had been co-cultured. Purified SCD-IP is a glycoprotein with an apparent molecular mass of 20 kDa. The molecular mass of the SCD-IP was estimated to be 18 kDa by mass spectrometry. Amino-terminal and two internal amino acid sequences of the pheromone revealed significant similarity to another Closterium pheromone, protoplast release-inducing protein (PR-IP) inducer of Closterium peracerosum-strigosum-littorale complex (C. pslc). These two pheromones induced different morphological reactions in each Closterium species. Based on these results, the diversity of sex pheromones is discussed.     

BIOCHEMICAL, PHYSIOLOGICAL, AND MOLECULAR ANALYSIS OF SEXUAL ISOLATION IN THE SPECIES COMPLEX CLOSTERIUM PERACEROSUM-STRIGOSUM-LITTORALE (CHLOROPHYTA)1

Closterium strains obtained from Japan ( NIES-64 and -65 ) and Nepal ( NIES-67 and -68 ) have been classified as the same taxonomic species; however, they are sexually isolated from each other. When NIES-64 and -65 cells were separately incubated in a medium in which both strains had previously been cultured together, release of protoplasts from both strains was observed. We suggest that factors responsible for the release of protoplasts from cells of both NIES-64 and -65 are produced in a mixed culture of these cells and function during conjugation. These factors, however, had no effect on the release of protoplasts from cells of strains NIFS-67 or -68. Alternatively, a protein that is responsible for the release of protoplasts from cells of NIES-68, called the protoplast-release-inducing protein ( PR-IP ), had no effect on the release of protoplasts from cells of strains NIES-64 or -65. When the media obtained from the culture of NIES-64 and -65 cells at various mixing ratios were analyzed by western blotting with antiserum to a 42-kDa subunit of PR-IP, no cross reaction was detected. In Southern hybridization analysis, no hybridizing band was observed when genomic DNAs of NIES-64 and -65 cells were probed with cDNAs encoding the two subunits of PR-IP. We suggest from these results that the factors responsible for the release of protoplasts from NIES-64 and -65 cells are not structurally similar to PR-IP. It is known that the release of PR-IP from NIES-67 cells can be induced by the action of another sex pheromone ( PR-IP inducer ) which is released by NIES-68 cells. In contrast, no protoplast-release-inducing activity was observed from either NIES-64 or -65 in a culture medium conditioned by opposite strains. We suggest that the conjugation systems employed by strains NIES-64/ NIES-65 and strains NIES-67 /NIES-68 differ, and we propose a possible mechanism of sexual isolation between these biological species .     

Purification and Characterization of a Novel Sex Pheromone that Induces the Release of Another Sex Pheromone during Sexual Reproduction of the Heterothallic Closterium peracerosum-strigosum-littorale Complex

A sex pheromone, PR-IP Inducer, which is released from mating-typeminus cells of Closterium, was purified by monitoring its biologicaleffect on the induction of the release of protoplast-release-inducingprotein (PR-IP) from mating-type plus cells. The purified PR-IPInducer had an apparent molecular mass of 22 kDa and of 18.7kDa as determined by SDS-PAGE and mass-spectrometric analysis,respectively. Staining with periodic acid-Schiff reagent indicatedthat PR-IP Inducer included a glycan chain. From the analysisof a dose-response curve, it seemed that PR-IP Inducer was ableto exert its activity over quite wide range of concentrations(1 x 10^–10–3 x 10^–7 M). It appears that PR-IPInducer is a novel glycoproteinaceous pheromone, as is PR-IP,and that it exerts its effect at the earliest stages of thesexual reproduction of the Closterium peracerosum-strigosum-littoralecomplex. ¹Recipient of a Fellowship for Japanese Junior Scientists fromthe Japan Society for the Promotion of Science.     

Detection and Evaluation of an Inducer of a Diffusible Mating Pheromone of the Heterothallic Closterium peracerosum-strigosum-littorale Complex

When mating-type plus cells of the Closterium peracerosum-strigosum-littoralecomplex were incubated in nitrogen-deficient medium obtainedfrom a 24-h-old mixed culture of mating-type plus and mating-typeminus cells, protoplast-release-inducing activity specific formating-type minus cells was detected in the medium. When mating-typeplus cells were incubated in the medium from a culture of exclusivelymating-type minus cells, protoplast-release-inducing activitywas also detected. These results suggested the existence ofa substance, released from mating-type minus cells, that hasthe ability to make mating-type plus cells release protoplast-release-inducingprotein (PR-IP). We designated it PR-IP Inducer. The PR-IP Inducerwas constitutively released from mt^– cells in the light.The PR-IP Inducer was heat-labile and had a relative molecularweight of 10,000 on gel filtration. We suggest that the PR-IPInducer is also a pheromonal substance that plays a role inthe initial events in the sexual communication of this Closteriumcomplex. (Received April 26, 1993; Accepted July 15, 1993)     

The sexual inducer of Volvox carteri: purification, chemical characterization and identification of its gene

The sexual inducer of Volvox carteri f. nagariensis is a glycoprotein and one of the most potent biological effector molecules known. It is synthesized by sperm cells and converts asexually growing males and females to the sexual pathway. Until now, large-scale production of the inducer was made impossible by an inherent biological `switch' mechanism, the spontaneous self-induction of asexually growing males. Here we describe a method overcoming this problem for the first time. Large-scale production and purification allowed a detailed chemical characterization of the inducer with respect to partial amino acid sequences and sugar composition. Chemically synthesized oligodeoxynucleotides corresponding to derived amino acid sequences were used to screen a genomic gene bank of V. carteri HK 10. A positive clone (Ind-28) was shown to encode the inducer gene by subcloning and sequencing.     

Analysis of binding of biotinylated protoplast-release-inducing protein that induces release of gametic protoplasts in the Closterium peracerosum-strigosum-littorale complex

A protoplast-release-inducing protein (PR-IP) which is released from mating-type plus (mt⁺) cells and induces the release of gametic protoplasts from matingtype minus (mt⁻) cells of Closterium was biotinylated and then used to examine the interaction of this protein with mt⁻ cells. The protoplast-release-inducing activity of PR-IP was not altered after the biotinylation. When mt⁻ cells that had been pre-cultured for 24 h were incubated with biotinylated PR-IP for 6 h in nitrogen-deficient medium that contained 1% (w/v) bovine serum albumin, and then washed with the same medium, only a 19-kDa polypeptide, the smaller subunit of PR-IP, was detected in cells by the avidin and biotinylated horseradish-peroxidase macromolecular complex system. The amount of bound 19-kDa polypeptide increased with increasing doses of PR-IP and reached a maximum at around 10 nM, reflecting the protoplast-release-inducing activity. From a Scatchard plot, the dissociation constant of the polypeptide was calculated to be 2.7 · 10⁻⁸ M. The binding of the polypeptide proceeded only after an appropriate period of pre-culture in the light, and the polypeptide was competitively displaced by non-biotinylated PR-IP. From these results, it appears that the PR-IP induces the release of protoplasts from mt⁻ cells by binding of a polypeptide of relative molecular mass 19000 to the receptor on the cell surface in a manner analogous to the binding of peptide hormones in animals.     

ANALYSIS OF GAMETIC PROTOPLAST RELEASE IN THE CLOSTERIUM PERACEROSUM-STRIGOSUM-LITTORALE COMPLEX (CHLOROPHYTA)1

A biologically active glycoprotein (protoplast-release-inducing protein; PR-IP), which induces the release of gametic protoplasts from mating type minus (mt^-) cells of the Closterium peracerosum-strigosum-littorale complex, was prepared from a medium in which mt^- and mt⁺ cells had been previously incubated together. The process of PR-IP-inducing protoplast release was analyzed. Induction of protoplast release was dependent upon the duration of both PR-IP treatment and preincubation in nitrogen-deficient mating medium before PR-IP treatment. Low cell density in the preculture stage had a significant stimulative effect upon the induction of protoplast release. Light was necessary for protoplast release, especially just before PR-IP treatment. Chloramphenicol and 3-(4-chlorophenyl)-1,1-dimethylurea (CMU) exerted inhibitory effects on protoplast release, especially when they were applied to the preculture stage but not when they were applied to the protoplast-releasing stage after the PR-IP treatment. We suggest that preculture at a low cell density under continuous light conditions that may cause metabolic changes in the chloroplast is a very important stage for gametic protoplast release in this Closterium.     

Pretreatment with CP-154526 blocks the modifying effects of alarm pheromone on components of sexual behavior in male, but not in female, rats

We previously demonstrated that an alarm pheromone released from male donor Wistar rats evoked several physiological and behavioral responses in recipient rats. However, the pheromone effects on social behavior were not analyzed. In the present study, we examined whether the alarm pheromone affects sexual behavior in male or female rats. When a pair of male and female subjects was exposed to the alarm pheromone during sexual behavior, the ejaculation latency was elongated, the number of mounts was increased, and the hit rate (number of intromissions/number of mounts and intromissions) was decreased in the male subject. In contrast, female sexual behavior was not affected by the alarm pheromone. When we exposed only the male or female subject of the pair to the pheromone just before sexual behavior, the results were similar: the pheromone effects were evident in male, but not in female, subjects. In addition, when we pretreated with corticotropin-releasing factor (CRF) antagonist (CP-154526) before exposing the male subject to the alarm pheromone, the pheromone effects were attenuated in a dose-dependent manner. These results indicate that the alarm pheromone modifies male, but not female, components of sexual behavior and that CRF participates in the effects.     

Inositol is specifically involved in the sexual program of the fission yeast Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe is a natural auxotroph for inositol and fails to grow in the complete absence of it. It was previously reported that a small concentration of inositol in the culture medium supports vegetative growth, but not mating and sporulation, and a tenfold of that concentration also supports mating and sporulation. The purpose of the present work was to investigate whether a moderate inositol starvation specifically affected events of the sexual program of development. A homothallic culture grown to the stationary phase in medium with a small inositol concentration was sterile but cells in the stationary phase of growth synchronously entered and completed the sexual cycle when inositol was added, without need of previous cell divisions. This suggests the involvement of inositol in a mechanism (or mechanisms) of the sexual program. The events of the program that were affected by inositol starvation were investigated. Commitment to mating and production of pheromone M were shown not to be inositol-dependent. A diploid strain homozygous at the mating-type locus and carrying a pat1-114 temperature-sensitive mutation in homozygous configuration sporulated under inositol starvation at the restrictive temperature; therefore starvation did not directly affect meiosis or sporulation. In contrast, production of pheromone P and the response of cells to pheromones were found to be inositol-dependent. The possibility that inositol or one of its derivative compounds is involved in pheromone P secretion and in pheromone signal reception is discussed.     

Roles for receptors, pheromones, G proteins, and mating type genes during sexual reproduction in Neurospora crassa

Here we characterize the relationship between the PRE-2 pheromone receptor and its ligand, CCG-4, and the general requirements for receptors, pheromones, G proteins, and mating type genes during fusion of opposite mating-type cells and sexual sporulation in the multicellular fungus Neurospora crassa. PRE-2 is highly expressed in mat a cells and is localized in male and female reproductive structures. Δpre-2 mat a females do not respond chemotropically to mat A males (conidia) or form mature fruiting bodies (perithecia) or meiotic progeny (ascospores). Strains with swapped identity due to heterologous expression of pre-2 or ccg-4 behave normally in crosses with opposite mating-type strains. Coexpression of pre-2 and ccg-4 in the mat A background leads to self-attraction and development of barren perithecia without ascospores. Further perithecial development is achieved by inactivation of Sad-1, a gene required for meiotic gene silencing. Findings from studies involving forced heterokaryons of opposite mating-type strains show that presence of one receptor and its compatible pheromone is necessary and sufficient for perithecial development and ascospore production. Taken together, the results demonstrate that although receptors and pheromones control sexual identity, the mating-type genes (mat A and mat a) must be in two different nuclei to allow meiosis and sexual sporulation to occur.     

Role of metabolism of the mating pheromone in sexual differentiation of the heterobasidiomycete Rhodosporidium toruloides.

A trypsin-type endopeptidase (Kamiya et al., Biochem. Biophys. Res. Commun. 94:855-860, 1980) responsible for the metabolism of rhodotorucine A, the farnesyl undecapeptide mating pheromone secreted by mating type A cells of Rhodosporidium toruloides, was biologically characterized. Metabolic activity was found to be present exclusively on the cell surface of the pheromone target cell. The activity was highly specific to the pheromone, and a biologically inactive analog which has the complete amino acid sequence of rhodotorucine A but lacks the farnesyl residue was not metabolized by intact cells. Pheromone metabolism was inhibited by trypsin substrates such as tosyl-L-arginine methyl ester. The presence of tosyl-L-arginine methyl ester strongly inhibited the sexual differentiation induced by the pheromone at a concentration which did not affect the vegetative growth of R. toruloides. Pheromone-induced sexual differentiation was also strongly inhibited by a metabolizable analog, rhodotorucine A S-oxide, but not by a non-metabolizable one. In mutants defective in early processes of mating, the decrease in the pheromone metabolic activity correlated well with the extent of loss of sensitivity to the pheromone. Both the pheromone metabolism and the capacity for sexual differentiation of a sterile mutant were restored concomitantly with reversion from the sterile to the fertile phenotype. These results suggested that metabolism of the mating pheromone plays an essential role in the process of sexual differentiation in R. toruloides.     

Variations in sexual dimorphism in the skulls of rats subjected to malnutrition, castration, and treatment with gonadal hormones

Two groups of weanling rats were subjected to malnutrition, one with periodic injections of testosterone (males) and the other with estradiol (females). Two other groups (castrated males or castrated females) received normal feedings. In control animals, the relative weights (mg/gm body weight) of testes, seminal vesicles, and ovaries were greater than in malnourished rats. However, relative weights of those organs in hormone-treated, malnourished animals were greater than in those subjected to malnutrition alone and still greater than in controls. Normal sexual cranial dimorphism (SCD) was decreased 16% by male castration, 23% by malnutrition, and 83% by estradiol treatment in malnourished females. On the other hand, normal SCD was increased 20% by female castration and more than 200% by testosterone treatment in malnourished males. All monosexual comparisons corroborated the bisexual range of distances found. Testicular but not ovarian secretions seemed to influence sexual cranial dimorphism. Malnutrition delayed SCD because of a deficiency of testosterone level in stressed males. It is suggested that estradiol in females may counteract sexual cranial development and that its inhibitory effect may be additive to the testosterone deficit evoked by malnutrition.     

Mating Effect on Sex Pheromone Production of the Oriental tobacco budworm,Helicoverpa assulta

This study was undertaken to clarify the suppression phenomenon of sex pheromone production after mating and its relationship to the physiological mechanism in adult females of Helicoverpa assulta, and determine the mating factor from males causing depletion of sex pheromonc production. Sex pheromone production of H. assulta females was mostly terminated in 3 hours after mating. Mated females maintained with a low titer of sex pheromone until 3 days when it started to increase again, which showed a characteristic of species mating more than once. The mated female again produced pheromone upon injection of pheromone biosynthesis activating neuropeptide (PBAN) or extracts of brain-suboesophageal ganglion complexes (Br-Sg) of mated female, which were shown similar pheromonotropic activities as compared with virgin females. These results indicated that the mating did not inhibit the receptivity of pheromone gland itself and PBAN biosynthesis in suboesophageal ganglion of the mated females. And it seems to support that the depletion of sex pheromone production is responsible for blocking of PBAN release from head. To investigate the mating factor from adult males, when extracts of reproductive organs of male were injected into hemocoel of virgin females evoking depletion of sex pheromone production as shown in mated female. The results suggest that a chemical substance(s) from the male reproductive organs could be responsible for the loss of sex pheromone biosynthesis in H. assulta.