Normal Mating Research Articles

Pneumocystis carinii cell wall biosynthesis kinase gene CBK1 is an environmentally responsive gene that complements cell wall defects of cbk-deficient yeast.

Pneumocystis species remain an important cause of life-threatening pneumonia in immunocompromised hosts, including those with AIDS. Responses of the organism to environmental cues both within the lung and elsewhere have been poorly defined. Herein, we report the identification of a cell wall biosynthesis kinase gene (CBK1) homologue in Pneumocystis carinii, isolated by differential display PCR, that is expressed optimally at physiological pH (7 to 8) as opposed to more acidic environments. Expression of Pneumocystis CBK1 was also induced by contact with lung epithelial cells and extracellular matrix. Translation of this gene revealed extensive homology to other fungal CBK1 kinases. Pneumocystis CBK1 expression was equal in the cyst and trophic life forms of the organisms. We further demonstrate that Pneumocystis CBK1 expressed in cbk1 Delta Saccharomyces cerevisiae cells restored defective cell wall separation during proliferation. Consistent with this, Pneumocystis CBK1 expression also stimulated transcription of the CTS1 chitinase in cbk1 Delta mutant yeast cells, an event necessary for cell wall separation. In addition, Pneumocystis CBK1 cDNA supported normal mating projection formation in response to alpha-factor in the cbk1 Delta yeast cells. Site-directed mutations of serine-303 and threonine-494, potential regulatory phosphorylation sites in Pneumocystis CBK1, abolished mating projection formation, indicating a role for these amino acid residues in CBK1 activity. These findings indicate that Pneumocystis CBK1 is an environmentally responsive gene that may function in signaling pathways necessary for cell growth and mating.

206POSTNATAL MANAGEMENT OF CHRYPTORCHID BANTENG CALVES CLONED BY NUCLEAR TRANSFER UTILIZING FROZEN FIBROBLAST CULTURES AND ENUCLEATED COW OVA

A multi-institutional collaboration was developed to evaluate the potential for conservation management of genetic variation in captive banteng calves via nuclear transfer cloning of a deceased genetically valuable individual. Fibroblast cultures frozen in 1978 were fused with enucleated cow ova and resulting embryos were transferred to domestic beef cattle for gestation. Two banteng (Bos javanicus) calves produced by heterologous nuclear transfer were delivered by standing Cesarean section on April 1 and 3, 2003. Birth weights for the two calves were 23.13 and 38.64kg, respectively. The larger calf failed to gain strength and died on April 8. The surviving calf was transferred from Sioux Center, IA, to the San Diego Wild Animal Park in Escondido, CA on April 18, 2003. The calf was offered a goat’s milk-based formula containing 21.9kJ metabolizable energy (ME) and 22% total solids. The total solids fraction consisted of 26.3% crude protein, 29.8% crude fat, 33.5% total carbohydrate. Intake and growth rate for this calf was compared to other hand-reared bovid calves produced by normal mating. Average daily gain from birth to 16 weeks of age was 767g day−1. A routine neonatal physical examination revealed that the surviving calf was bilaterally cryptorchid. The large calf that did not survive was noted to have one abdominally retained testicle, whereas the other testicle was scrotal. Ultrasound examination of the healthy banteng calf confirmed abdominal localization of the retained testicles. Both testes were located in the abdominal cavity about 4–6cm from the caudal pole of the kidneys. Therapeutic intervention was considered because of the genetic value of the calf and because the cell donor exhibited normal scrotal testicles. In July, 2003, orchipexy was conducted on this calf. The left testis was exteriorized through a small abdominal flank incision. After freeing it from tissue holding it in place, it was relocated through a subcutaneous tunnel to a pouch created just in front of the scrotum. The attachments to the testis were not long enough to allow the testis to be placed fully in the scrotum. At a follow-up examination conducted four weeks after the surgery, the left testicle was palpable in the subcutaneous pouch just cranial to the scrotum. Under ultrasound examination, the left testicle was easily identified less than 1 cm deep with echogenic texture of a testis. Color flow Doppler ultrasound analysis demonstrated pulsatile flow in the center of the testis synchronous with the heart beat. These findings indicate testicular viability and blood supply.

Variable Female Mating Positions and Offspring Sex Ratio in the Spider Pityohyphantes phrygianus (Araneae: Linyphiidae)

Chromosomal sex determination and male heterogamety have been thought to seriously impede direct sex ratio control. However, in Pityohyphantes phrygianus, a solitary sheetweb spider with a skewed sex ratio, earlier experimental studies suggested that there are options for female control of offspring sex ratio, if females change their position during the normal mating sequence. Here we show that under natural conditions there is considerable between-female variation in positions, especially after termination of mating. Computer simulations of the orientation of female inner genitalia suggest that sperm are placed in different storage sites depending on the positions adopted. This means that a specific position after mating might potentially influence offspring sex ratio. The variance in offspring sex ratio among females in earlier experiments was binomially distributed, which leads us to conclude that females control the mean sex ratio but do not exercise direct control of the sex of individual offspring.

Induction of infertility in adult male bonnet monkeys by immunization with phage-expressed peptides of the extracellular domain of FSH receptor

Active immunization of proven fertile adult male bonnet monkeys ( Macaca radiata) with phage-expressed follicle-stimulating hormone receptor (FSHR)-specific peptides from the extracellular domain resulted in a progressive drop in sperm count with all animals becoming azoospermic by day 100. However, serum testosterone concentrations were unaltered during the entire course of study and animals exhibited normal mating behaviour. Breeding studies with proven fertile female monkeys revealed that all the immunized males were infertile. Following interruption of immunization on day 225, sperm counts returned to normal with restoration of fertility. These results indicate that infertility can be induced in adult male monkeys by interfering with the action of FSH using specific peptides of the extracellular domain of FSHR as antigens, without the risk of producing cross-reacting antibodies to the other glycoprotein hormones.

The MPN mag: introducing a critical area mediating pheromonal and hormonal regulation of male sexual behavior.

Mating behavior in male hamsters is regulated by a chemosensory pathway that converges on the bed nucleus of the stria terminalis (BST) and the medial nucleus of the amygdala (Me). Both the BST and the Me project to the lateral part of the medial preoptic area. Lesion studies have identified a small group of large cells referred to as the magnocellular medial preoptic nucleus (MPN mag) whose integrity is required for normal mating behavior. Our data, summarized within, indicate that the MPN mag is a sexually differentiated nucleus in a large steroid-responsive network that relays pheromonal signals from the sensory systems to the motor areas to affect behavior.

Epididymal dysfunction initiated by the expression of simian virus 40 T-antigen leads to angulated sperm flagella and infertility in transgenic mice.

We have generated two transgenic mouse lines (GPX5-Tag1 and GPX5-Tag2) by expressing the Simian virus 40 large and small T-antigens under a 5-kb promoter of the murine glutathione peroxidase 5 (GPX5) gene. In GPX5-Tag1 mice, with a high level of T-antigen expression, severe dysplasia was found in the epididymis and seminal vesicles. These mice also developed adrenal and prostate tumors, and spermatogenesis was disrupted. In GPX5-Tag2 mice, with a lower level of T-antigen expression, the only histological change was the slightly hyperplastic epithelium in the initial segment of the epididymidis and in the seminal vesicles. Despite normal mating behavior, these mice were infertile. The most conspicuous feature of the sperm was angulation of the flagellum, which appeared during epididymal transit, probably due to the observed reduction in the osmotic pressure of cauda epididymidal fluid. The angulation did not affect the motility or kinematic parameters of the sperm, but the sperm were also incapable of fertilization in vitro. The lack of expression of several genes specific for the initial segment suggests that in the GPX5-Tag2 mice the transgene expression brings about a differentiation arrest in this part of epididymis. This novel mouse line provides a model for epididymal dysfunction leading to defects in posttesticular sperm maturation and infertility.

Effects of advanced maternal age on development of preimplantation mouse embryos obtained from delayed mating

Rodent embryos resulting from delayed mating grow more quickly than those resulting from normal mating. To clarify the effects of maternal age on this enhanced growth and rapid differentiation of mouse embryos obtained from delayed mating, we studied normal (0 h after ovulation) and delayed mating (6 h after ovulation) subgroups in two different age groups (middle-aged, 9–11 months; elderly, 13–15 months), and compared the number of cells and the proportion of blastocyst formed (morphologically advanced blastocysts) among them. In middle-aged female mice, preimplantation embryos derived from delayed mating progressed more rapidly than their normally mated counterparts. The number of cells and the proportion of blastocyst formed among embryos from delayed mating were almost 3 h ahead of those of normal embryos. In the delayed mating subgroup from elderly mice, an enhanced rate of development was not observed. There was no difference in development of normally mated embryos from middle-aged females compared to each counterpart from elderly female mice. Preimplantation mouse embryonic development from delayed mating may be affected by advanced maternal age.

Immune investment and sperm competition in a beetle

AbstractIndividuals that invest more in immunity may not be able to invest as much in o\ther life history traits. The overall effects on fitness depend on the balance of investment in life history traits and unnecessary investment in immunity may lower fitness. Adult mealworm beetles (Tenebrio molitor L.) modulate their investment according to the perceived risk of infection as larvae; the amount of investment can be assessed by body coloration. This prophylactic investment in immunity can be used to assess the costs of investment when no immune challenge is present. Whether investment in immunity is traded off against sperm competitive ability, another important fitness trait in insects, was investigated. Males that had invested more in immunity (dark males) competed against males that had invested less (light males) for fertilization of offspring. Dark males did lose sperm precedence over time, whereas light males did not. However, this decrease in sperm offensive ability may not result in decreased fitness for darker males under normal female mating frequencies; the decrease in offspring did not occur for 1 week, but females that have constant access to males mate once a day, which would negate any long‐term effects of male mating order. Thus, prophylactic investment in immunity does not produce immediate reductions in a male's ability to gain fertilizations. The costs to immune investment may be born by other fitness traits in T. molitor.

Mutation of a novel gene results in abnormal development of spermatid flagella, loss of intermale aggression and reduced body fat in mice.

ROSA22 male mice are sterile due to a recessive gene-trap mutation that affects development of the spermatid flagellum. The defect involves the flagellar axoneme, which becomes unstable around the time of its assembly. Despite a subsequent complete failure in flagellar assembly, development of the spermatid head appears normal and the spermatid head is released at the correct stage in spermatogenesis. The mutation is pleiotropic. Although ROSA22 homozygote males have normal levels of circulating testosterone and display normal mating behavior, they do not exhibit intermale aggressive behavior and have reduced body fat. The mutated gene (Gtrgeo22) maps to mouse chromosome 10 and is closely flanked by two known genes, Madcam1 and Cdc34. Ribonuclease protection analysis indicates that expression of the flanking genes is unaffected by the mutation. Gtrgeo22 is expressed at low levels in epithelial cells in several tissues, as well as in testis and brain. Analysis of the peptide coding sequence suggests that Gtrgeo22 encodes a novel transmembrane protein, which contains dileucine and tyrosine-based motifs involved in intracellular sorting of transmembrane proteins. Analysis of the Gtrgeo22 gene product should provide novel insight into the molecular basis for intermale aggression and sperm flagellar development.

Identification of the cAD1 sex pheromone precursor in Enterococcus faecalis.

The Enterococcus faecalis virulence plasmid pAD1 encodes a mating response induced by exposure to an octapeptide sex pheromone, cAD1, secreted by plasmid-free enterococci. The determinant for the pheromone in E. faecalis FA2-2, designated cad, was found to encode a 309-amino-acid lipoprotein precursor with the last 8 residues of its 22-amino acid signal sequence representing the cAD1 moiety. The lipoprotein moiety contained two 77-amino-acid repeats (70% identity) separated by 45 residues. The nonisogenic E. faecalis strain V583 determinant encodes a homologous precursor protein, but it differs at two amino acid positions, both of which are located within the pheromone peptide moiety (positions 2 and 8). Construction of a variant of strain FA2-2 containing the differences present in V583 resulted in cells that did not produce detectable cAD1. The mutant appeared normal under laboratory growth conditions, and while significantly reduced in recipient potential, when carrying pAD1 it exhibited a normal mating response. A mutant of FA2-2 with a truncated lipoprotein moiety appeared normal with respect to recipient potential and, when carrying plasmid DNA, donor potential. A gene encoding a protein designated Eep, believed to be a zinc metalloprotease, had been previously identified as required for pheromone biosynthesis and was believed to be involved in the processing of a pheromone precursor. Our new observation that the pAD1-encoded inhibitor peptide, iAD1, whose precursor is itself a signal sequence, is also dependent on Eep is consistent with the likelihood that such processing occurs at the amino terminus of the cAD1 moiety.

Factors involved in the post-copulatory neural inhibition of pheromone production in Choristoneura fumiferana and C. rosaceana females

Normal mating lasts approximately 3 h in Choristoneura fumiferana and C. rosaceana. Data generated from interrupted matings showed that the act of mating did not suppress pheromone production (pheromonostasis) in either species although, in C. rosaceana, pheromone titre declined slightly the night following mating. In both species the migration of sperm to the spermatheca (SP) occurred several hours after mating, and coincided with a significant and permanent depression in pheromone titre, as well as egg fertilisation and oviposition. However, disrupting matings within 2 h of the onset resulted in oviposition patterns similar to virgins in both species, with mostly infertile eggs being laid by C. fumiferana females while oviposition was totally inhibited in C. rosaceana. The transection of the ventral nerve cord (VNC) 1 h post-mating did not result in the depression of pheromone titres the following night in either species but if the VNC was transected 3 h post-mating, pheromonostasis was observed. While 25% of C. fumiferana females had sperm in their SP 2 h after mating, it took at least 4 h in C. rosaceana. This suggests that while the physical presence of sperm in the SP may play some role in the termination of pheromone production in C. fumiferana, other factors must trigger the neural signal that elicits pheromonostasis in both species. A better understanding of the temporal dynamics of both apyrene and eupyrene sperm within the different parts of the female reproductive system might clarify these interspecific differences.

Methylation Is Involved in the Ustilago maydis Mating Response

Methionine auxotrophs of Ustilago maydis were deficient in mating; unlike wild-type cells, they neither induced nor produced normal mating filaments in the presence of compatible cells. The deficiency was most severe when cells were located some distance apart, but when in direct contact with compatible cells methionine auxotrophs mated and infected plants fairly normally. The mating deficiency was genetically linked to the methionine auxotrophy, segregating with it through in planta crosses. Wild-type cells exposed to the methyltransferase inhibitors ethionine and homocysteine thiolactone were similarly impaired in mating. Exogneous methionine, S-adenosylmethionine (SAM), synthetic mating pheromone, or cAMP all compensated for the mating impairment of the auxotrophs to some extent. Although SAM-dependent methylation could influence activities of various molecules in diverse pathways, these observations indicate that the most likely cause of the mating deficiency in met− cells is failure to methylate a component of the U. maydis pheromone signal transduction pathway.

Oestrogen at the neonatal stage is critical for the reproductive ability of male mice as revealed by supplementation with 17beta-oestradiol to aromatase gene (Cyp19) knockout mice.

Aromatase P450 (CYP19) is an enzyme responsible for the conversion of androgens to oestrogens. We generated CYP19 knockout (ArKO) mice by targeted disruption of Cyp19 and studied the role of oestrogens in male reproductive ability. Approximately 85% of ArKO males were unable to sire offspring. However, no obvious difference was found in testicular and epididymal weights, numbers of sperm in the epididymis or the ability of sperm to fertilize eggs in vitro between wild-type and ArKO males. An examination of mating behaviour demonstrated that ArKO males showed an impairment in mounting behaviour against sexually mature females. The inability of more than 90% of ArKO males to sire offspring was reversed by repeated subcutaneous injections of 17beta-oestradiol when initiated on the day of birth. The effects of 17beta-oestradiol on reproduction were concentration dependent and evident when supplementation was initiated on day 7, but not on day 15 after birth. These findings suggest that oestrogens acting during neonatal life are required for normal mating behaviour in adulthood.

Male sexual dysfunction in mice bearing targeted mutant alleles of the PEA3 ets gene.

PEA3, a member of the Ets family of transcriptional regulatory proteins, is expressed in a unique spatial and temporal pattern during mouse embryogenesis; its overexpression is positively correlated with HER2-mediated breast tumorigenesis in both humans and mice. To determine whether PEA3 plays a part in development and oncogenesis and to uncover its normal physiological role, we generated mice lacking functional PEA3 by gene targeting in embryonic stem cells. PEA3(-/-) mice arose from heterozygous crosses with the expected Mendelian frequency, revealing that PEA3 is dispensable for embryogenesis. PEA3 mutant mice displayed no overt phenotype and lived a normal life span. However, PEA3-deficient males failed to reproduce. PEA3 is expressed in several male sexual organs, but gross and histological analyses of the organs from PEA3(-/-) mice revealed no abnormalities. Spermatogenesis and spermiogenesis also appeared normal in mice homozygous for the PEA3 mutation, and their sperm were capable of fertilizing eggs in vitro. PEA3(-/-) males engaged in normal mating behavior, but they did not set copulatory plugs and sperm could not be detected in the uteri of females that had mated with PEA3(-/-) males. Erections could be evoked by abdominal pressure in PEA3-deficient male mice, and the results of in vitro experiments revealed that the corpus cavernosum isolated from PEA3 mutant males relaxed in response to acetylcholine. Therefore, the infertility of PEA3 mutant males involves either mechanisms proximal to the cavernosal smooth muscle or an ejaculatory dysfunction. However, PEA3 mutant mice are phenotypically distinguishable from other knockout mice with such deficits and thus provide a unique model for further investigation of male sexual dysfunction.

The “low-dose” concept and the paradoxical effects of prolactin on grooming and sexual behavior

The effects of prolactin on animal behavior include the stimulation of novelty-induced grooming in rats. This effect has been demonstrated in hyperprolactinaemic animals bearing pituitary homografts under the kidney capsule or after intracerebroventricular (i.c.v.) administration of prolactin. Since plasma prolactin levels in hyperprolactinaemic rats are similar to those of animals injected with low doses of rat prolactin, we studied the effects of this hormone injected subcutaneously (s.c.) in a dose range of 5–50 μg/kg. Novelty-induced grooming was enhanced only in rats injected with 5 or 10 μg/kg rat prolactin, whereas no effect was observed after the s.c. injection of the higher dose. The sexual behavior of male rats is also affected by prolactin. Male rats with normal mating activity showed enhanced sexual behavior when injected s.c. with rat prolactin (5, 10 or 50 μg/kg). In animals with poor sexual performance or in impotent rats, prolactin (5 or 10 μg/kg, but not 50 μg/kg) restored the full pattern of sexual behavior. An increased lordosis quotient was also observed in ovariectomized rats treated with prolactin 5 or 10 μg/kg. These results suggest that, besides the duration of hyperprolactinaemia, the effective level of plasma prolactin is important for the expression of the behavioral effects of this hormone.

Correlation of Photosynthetic Products of Symbiotic Chlorella with the Mating Reactivity Rhythms in a Mutant Strain of Paramecium bursaria

Paramecium bursaria shows many kinds of circadian rhythms, including a mating reactivity rhythm. P. bursaria cells normally contain several hundred Chlorella in the cytoplasm as endosymbionts. We found an interesting mutant strain (Ok2) from nature. Chlorella-contain green cells (Ok2) showed a normal mating reactivity rhythm in a constant light condition (LL) or constant darkness (DD). However, Chlorella-free white cells (Ok2w) derived from Ok2 did not display a mating reactivity rhythm in LL. In DD, they showed a normal mating reactivity rhythm. When Ok2w cells were infected with Chlorella isolated from green cells that show normal mating reactivity rhythms, they exhibited a circadian mating rhythm in LL. The green Ok2 cells reverted to the non-reactive state in LL by treatment with the herbicide paraquat. Sugar components in the cytosol of Ok2 and Ok2w were analyzed by HPLC, and four kinds of sugars were identified in Ok2 cells of day time. When maltose and maltotriose were added to Ok2w cell culture, the mating reactivity rhythms appeared in white cells in LL. These results suggest that the photosynthetic products of symbiotic Chlorella are closely related to the expression of circadian rhythms in a mutant strain of P. bursaria.

Conversion of Testosterone to Estradiol May Not Be Necessary for the Expression of Mating Behavior in Male Syrian Hamsters (Mesocricetus auratus)

Male sexual behavior is mediated in part by androgens, but in several species, mating is also influenced by estradiol formed locally in the brain by the aromatization of testosterone. The role of testosterone aromatization in the copulatory behavior of male Syrian hamsters is unclear because prior studies are equivocal. Therefore, the present study tested whether blocking the conversion of testosterone to estradiol would inhibit male hamster sexual behavior. Chronic systemic administration of the nonsteroidal aromatase inhibitor Fadrozole (2.0 mg/kg/day) for 5 or 8 weeks did not significantly increase mount latency or reduce mount frequency, intromission frequency, ejaculation frequency, or anogenital investigation relative to levels shown by surgical controls. However, Fadrozole effectively inhibited aromatase activity, as evidenced by the suppression of estrogen-dependent progesterone receptor immunoreactivity in the male hamster brain. The JZB39 anti-progesterone receptor antibody labeled significantly more neurons in brains of sham-treated hamsters than in brains of Fadrozole-treated hamsters. These data suggest that aromatization of testosterone to estradiol is not necessary for normal mating behavior in Syrian hamsters.

Effect of chronic administration of an aromatase inhibitor to adult male rats on pituitary and testicular function and fertility.

The aim of the present study was to evaluate the effects of the administration of a potent non-steroidal aromatase inhibitor, anastrozole, on male reproductive function in adult rats. As anastrozole was to be administered via the drinking water, a preliminary study was undertaken in female rats and showed that this route of administration was effective in causing a major decrease in uterine weight (P<0.02). In an initial study in male adult rats, anastrozole (100 mg/l or 400 mg/l) was administered via the drinking water for a period of 9 weeks. Treatment with either dose resulted in a significant increase ( approximately 10%) in testis weight and increase in plasma FSH concentrations (P<0.01) throughout the 9 weeks. Mating was altered in both groups of anastrozole-treated rats, as they failed to produce copulatory plugs. Histological evaluation of the testes from anastrozole-treated rats revealed that spermatogenesis was grossly normal. In a more detailed study, adult rats were treated with 200 mg/l anastrozole via the drinking water for periods ranging from 2 weeks to 1 year. Plasma FSH and testosterone concentrations were increased significantly (P<0.001) during the first 19 weeks of treatment. However, LH concentrations were increased only at 19 weeks (P<0.001) in anastrozole-treated rats, and this coincided with a further increase in circulating and intratesticular testosterone concentrations (P<0.05). No consistent change in inhibin-B concentrations was observed during the study. Suppression of plasma oestradiol concentrations could not be demonstrated in anastrozole-treated animals, but oestradiol concentrations in testicular interstitial fluid were reduced by 18% (P<0.01). Mating was again inhibited by anastrozole treatment, but could be restored by s.c. injection of oestrogen, enabling demonstration that rats treated for 10 weeks or 9 months were still fertile. Testis weight was increased by 19% and 6% after treatment for 19 weeks and 1 year, respectively. Body weight was significantly decreased (P<0.01) by 19 weeks of anastrozole treatment; after 1 year the animals appeared to have less fat as indicated by a 27% decrease in the weight of the gonadal fat pad. The majority of anastrozole-treated animals had testes with normal spermatogenesis but, occasionally, seminiferous tubules showed abnormal loss of germ cells or contained only Sertoli cells. Ten percent of anastrozole-treated animals had testes that appeared to contain only Sertoli cells, and one rat had 'giant' testes in which the tubule lumens were severely dilated. Morphometric analysis of the normal testes at 19 weeks showed no difference in the number of Sertoli cells or germ cells, or the percentage volumes of the seminiferous epithelium, tubule lumens and interstitium between control and anastrozole-treated rats. On the basis of the present findings, oestrogen appears to be involved in the regulation of FSH secretion and testosterone production, and is also essential for normal mating behaviour in male rats. Furthermore, these data suggest that the brain and the hypothalamo-pituitary axis are considerably more susceptible than is the testis to the effects of an aromatase inhibitor. Anastrozole treatment has resulted in a model of brain oestrogen insufficiency.

Isolating aging mutants: a novel method yields three strains of the nematode Caenorhabditis elegans with extended life spans

We designed a novel procedure for the isolation of mutant strains with significantly increased life spans in the nematode Caenorhabditis elegans. This procedure involves using heat-shock to screen a large number of animals and isolate a few which are more resistant to heat-shock stress. From the heat-shock-resistant animals, three mutant strains, HG25, HG96, and HG246, all exhibiting increased life span, were isolated. One mutant strain (HG246) develops more slowly than the wild-type strain, N2. Two mutant strains, HG96 and HG246, exhibit lower fertility than the wild-type. Each of the three mutant strains has a normal appearance. Their locomotive behavior also appears normal; only HG246 shows slightly slower movement. Their feeding behavior appears normal, and the males of HG25 and HG96 show normal mating behavior. However, the males of HG246, either are defective in their mating ability or their sperm are defective. The results indicate that heat-shock can be used as a means to facilitate the isolation of mutants which have longer life expectancy.

Direct binding and In vivo regulation of the fission yeast p21-activated kinase shk1 by the SH3 domain protein scd2.

The Ste20/p21-activated kinase homolog Shk1 is essential for viability and required for normal morphology, mating, and cell cycle control in the fission yeast Schizosaccharomyces pombe. Shk1 is regulated by the p21 G protein Cdc42, which has been shown to form a complex with the SH3 domain protein Scd2 (also called Ral3). In this study, we investigated whether Scd2 plays a role in regulating Shk1 function. We found that recombinant Scd2 and Shk1 interact directly in vitro and that they interact in vivo, as determined by the two-hybrid assay and genetic analyses in fission yeast. The second of two N-terminal SH3 domains of Scd2 is both necessary and sufficient for interaction with Shk1. While full-length Scd2 interacted with only the R1 N-terminal regulatory subdomain of Shk1, a C-terminal deletion mutant of Scd2 interacted with both the R1 and R3 subdomains of Shk1, suggesting that the non-SH3 C-terminal domain of Scd2 may be involved in defining specificity in SH3 binding domain recognition. Overexpression of Scd2 stimulated the autophosphorylation activity of wild-type Shk1 in fission yeast but, consistent with results of genetic analyses, did not stimulate the activity of a Shk1 protein lacking the R1 subdomain. Results of additional two-hybrid experiments suggest that Scd2 may stimulate Shk1 catalytic function, at least in part, by positively modulating protein-protein interaction between Cdc42 and Shk1. We propose that Scd2 functions as an organizing center, or scaffold, for the Cdc42 complex in fission yeast and that it acts in concert with Cdc42 to positively regulate Shk1 function.