Acp36DE is required for uterine conformational changes in mated Drosophila females

In a multitude of animals with internal fertilization, including insects and mammals, sperm are stored within a female''s reproductive tract after mating. Defects in the process of sperm storage drastically reduce reproductive success. In Drosophila males, “Acp” seminal proteins alter female postmating physiology and behavior, and are necessary for several aspects of sperm storage. For example, Acps cause a series of conformational changes in the mated female''s reproductive tract that occur during and immediately after mating. These conformational changes have been hypothesized to aid both in the movement of sperm within the female and in the subsequent storage of those sperm. We used RNAi to systematically knock down several Acps involved in sperm storage to determine whether they played a role in the mating-induced uterine conformational changes. Mates of males lacking the glycoprotein Acp36DE, which is needed for the accumulation of sperm in the storage organs, fail to complete the full sequence of the conformational changes. Our results show that uterine conformational changes are important for proper accumulation of sperm in storage and identify a seminal protein that mediates these changes. Four Acps included in this study, previously shown to affect sperm release from storage (CG9997, CG1656, CG1652, and CG17575), are not necessary for uterine conformational changes to occur. Rather, consistent with their role in later steps of sperm storage, we show here that their presence can affect the outcome of sperm competition situations.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.     

From the Cover: Integrated 3D view of postmating responses by the Drosophila melanogaster female reproductive tract,obtained by micro-computed tomography scanning

Physiological changes in females during and after mating are triggered by seminal fluid components in conjunction with female-derived molecules. In insects, these changes include increased egg production, storage of sperm, and changes in muscle contraction within the reproductive tract (RT). Such postmating changes have been studied in dissected RT tissues, but understanding their coordination in vivo requires a holistic view of the tissues and their interrelationships. Here, we used high-resolution, multiscale micro-computed tomography (CT) scans to visualize and measure postmating changes in situ in the Drosophila female RT before, during, and after mating. These studies reveal previously unidentified dynamic changes in the conformation of the female RT that occur after mating. Our results also reveal how the reproductive organs temporally shift in concert within the confines of the abdomen. For example, we observed chiral loops in the uterus and in the upper common oviduct that relax and constrict throughout sperm storage and egg movement. We found that specific seminal fluid proteins or female secretions mediate some of the postmating changes in morphology. The morphological movements, in turn, can cause further changes due to the connections among organs. In addition, we observed apparent copulatory damage to the female intima, suggesting a mechanism for entry of seminal proteins, or other exogenous components, into the female’s circulatory system. The 3D reconstructions provided by high-resolution micro-CT scans reveal how male and female molecules and anatomy interface to carry out and coordinate mating-dependent changes in the female’s reproductive physiology.Mating induces physiological changes in females that enhance fertility. Many of these changes result from the transfer of seminal fluid, a complex chemical mixture containing sperm, male-derived molecules, and membranous vesicles (1–6), acting in the context of, and in conjunction with, female molecules and physiology. In insects and mammals, examples include seminal components that induce contraction of the female reproductive tract (RT) musculature (7–10), stimulate ovulation (11–13), regulate semen coagulation (14, 15), aid sperm movement and storage in the female RT (16, 17), and modulate female immune responses postmating (18, 19).Drosophila melanogaster is a powerful system with which to dissect how male-derived molecules interact with female physiology to cause the cascade of postmating responses required for fertility. Drosophila males transfer >200 different seminal fluid proteins (SFPs) to females during mating (19–21). Within the female RT, SFPs induce a series of conformational changes of the uterus (16, 22). Specific SFPs increase ovulation and oviposition rates (12, 13, 23), promote sperm storage (16, 24, 25), increase feeding (26), decrease intestinal transit rates (27, 28), and increase synaptic development at female RT neuromuscular junctions (10). Although much is known about the molecules and interactions that mediate these female postmating responses, next to nothing is known about the physical mechanics of the postmating changes and their response to male and female molecules.The physical act of mating, SFPs, and sperm receipt each modulate the release of signaling molecules in the Drosophila female RT (29). The Drosophila female RT is a complex system whose subregions differ in function and in the release and reuptake of signaling molecules; each region has a characteristic temporal and spatial identity postmating. Thus, postmating physiological changes are not simply a sequence of independent events but rather a coordinated process required for optimal fertility. Previous studies carried out on dissected tissues or specimens did not allow observation of the interactions and coordination among RT organs in situ within mating, or mated, flies.To visualize spatially the coordinated and concurrent organ rearrangements within the confines of the female abdomen during insemination through to egg laying, we used high-resolution, multiscale micro-computed tomography (CT) scans. These scans give a holistic 3D view of the interrelationship among reproductive organs in situ, as females respond to mating and its chemical components. With views ranging from 400-nm to 4-μm voxel sizes of the interior of female flies before, during, and at several times after mating, we observed looping, opening/closing, and repositioning of RT organs within the female. These data allow us to revisit, and newly define, effects of male and female molecules on female postmating physiology.     

Evolutionary EST analysis identifies rapidly evolving male reproductive proteins in Drosophila

Sequence comparisons of genomes or expressed sequence tags (ESTs) from related organisms provide insight into functional conservation and diversification. We compare the sequences of ESTs from the male accessory gland of Drosophila simulans to their orthologs in its close relative Drosophila melanogaster, and demonstrate rapid divergence of many of these reproductive genes. Nineteen ( approximately 11%) of 176 independent genes identified in the EST screen contain protein-coding regions with an excess of nonsynonymous over synonymous changes, suggesting that their divergence has been accelerated by positive Darwinian selection. Genes that encode putative accessory gland-specific seminal fluid proteins had a significantly elevated level of nonsynonymous substitution relative to nonaccessory gland-specific genes. With the 57 new accessory gland genes reported here, we predict that approximately 90% of the male accessory gland genes have been identified. The evolutionary EST approach applied here to identify putative targets of adaptive evolution is readily applicable to other tissues and organisms.     

Two cleavage products of the Drosophila accessory gland protein ovulin can independently induce ovulation

Proteins and peptides in Drosophila melanogaster seminal fluid induce mated females to increase their rates of egg deposition. One seminal-fluid protein, ovulin (Acp26Aa), stimulates an early step in the egg-laying process, the release of oocytes by the ovary. Ovulin, upon transfer to females, is cleaved sequentially within the mated female's reproductive tract. Here, we show that systemic ectopic expression of ovulin is sufficient to stimulate ovulation in unmated females. By using this assay to assess the functionality of ovulin's cleavage products, we find that two of the four cleavage products of ovulin can stimulate ovulation independently. Thus, ovulin's cleavage in mated females is not destructive and instead may liberate additional functional products with potential to modulate ovulation independently.     

A decade of the sperm‐washing programme: correlation between markers of HIV and seminal parameters

Objectives

The aim of the study was to use a decade of experience of sperm washing to assess the effect of HIV disease on semen parameters and to highlight the continuing importance of risk reduction when some controversially advocate the safety of timed unprotected intercourse for conception in the ‘stable’ HIV‐positive man.

Methods

Semen parameters of 439 fresh samples used for sperm washing/intrauterine insemination (IUI) were correlated against markers of HIV disease [CD4 cell count, viral load (VL), duration of HIV infection and use of antiretroviral therapy] and the risk of detectable virus in semen was assessed.

Results

A significant positive correlation was observed between CD4 cell count and total sperm count, progressive motility, post‐preparation/insemination concentration, progressive motility and total motile count inseminated (TMCI), and a significant negative correlation was observed between CD4 cell count and normal sperm morphology (Spearman's correlation; P<0.05). There was no significant difference in any parameter between samples in which VL was detectable and those in which it was undetectable. The use of highly active antiretroviral therapy (HAART) significantly decreased total sperm count, progressive motility, post‐preparation count and TMCI and significantly increased proportion of abnormal forms (Mann–Whitney tests; P<0.05). There was a significant negative correlation between duration of HAART use and concentration, total sperm count and post‐preparation motility and between years since diagnosis and post‐preparation motility. In 9.7% of IUI cycles performed with fresh sperm in men on HAART with undetectable VL, detectable HIV was found in either pre‐ or post‐wash seminal samples.

Conclusion

Our data suggest a negative effect of low CD4 cell count and the use of HAART on semen. The significant proportion of ‘stable’; men with undetectable serum VL but virus in semen confirms the continued importance of such risk reduction.     

Longevity and the stress response in Drosophila

The concept that lifespan is a function of the capacity to withstand extrinsic stress is very old. In concordance with this, long-lived individuals often have increased resistance against a variety of stresses throughout life. Genes underlying the stress response may therefore have the ability to affect lifespan. The progress in modern genetic techniques has allowed researchers to test this idea. The general stress response involves the expression of stress proteins, such as chaperones and antioxidative proteins, downregulation of genes involved in energy metabolism and the release of protective substances. Do these same changes in patterns of expression have the ability to mitigate ageing and prolong lifespan? It appears that parts of this response indeed are also associated with extended longevity, whereas some elements are not, due to their high cost or long-term deleterious consequences. Here we briefly review the state of the art of research on ageing and longevity in the model organism Drosophila, with focus on the role of the general stress response. We will conclude by contemplating some of the implications of the findings in this research and will suggest several directions for future research.     

Protein-specific manipulation of ejaculate composition in response to female mating status in Drosophila melanogaster

Female promiscuity can generate postcopulatory competition among males, but it also provides the opportunity for exploitation of rival male ejaculates. For example, in many insect species, male seminal fluid proteins (Sfps) transferred in a female's first mating stimulate increased fecundity and decreased receptivity to remating. Subsequent mates of females could potentially take advantage of the effects of the first male's Sfps and strategically reduce investment in their own ejaculate. We compared postmating responses (fecundity and sexual receptivity) of Drosophila melanogaster females after their first (virgin) matings (V), to the responses of females remating (M) 24 h after their first mating. The results show that M matings fail to boost fecundity and, thus, males are unlikely to gain fitness from transferring Sfps whose sole function-in V matings-is fecundity-stimulation. However, males can protect their likelihood of paternity in M matings through the transfer of receptivity-inhibiting Sfps. The levels of a fecundity-stimulating Sfp (ovulin) were significantly lower in M females relative to V females, at the same time point shortly after the end of mating. In contrast, the levels of a key receptivity-inhibiting Sfp (sex peptide) were the same in M and V females. These results support the hypothesis that males can adaptively tailor the composition of proteins in the ejaculate, allowing a male to take advantage of the fecundity-stimulating effects of the previous male's ovulin, yet maintaining investment in sex peptide. Furthermore, our results demonstrate sophisticated protein-specific ejaculate manipulation.     

The presence of androgen-binding protein in the guinea-pig testis, epididymis and epididymal fluid

Androgen-binding protein (ABP) is present in the guinea-pig testis, epididymis and epididymal fluid. Guinea-pig ABP sediments as an approx. 4.6S species on sucrose gradients containing 0.01 M KCl. Electrophoresis on non-denaturing polyacrylamide gels indicated that specific androgen binding was present in epididymal cytosol, but not in plasma. Time-course studies indicated that binding equilibrium is approached in about 2.5 h; the dissociation half-time of [3H]5 alpha-DHT from guinea-pig ABP is 5.64 +/- 0.62 h (n = 6) at 4 degrees C. The relative affinities of some steroids for guinea-pig ABP in relation to 5 alpha-DHT = 1 are: testosterone = 0.55 +/- 0.13 (n = 4), estradiol = 0.14 +/- 0.03 (n = 4), the anti-androgen cyproterone acetate = 0.0025 +/- 0.0002 (n = 3). Guinea-pig ABP exhibited an equilibrium dissociation constant of 6.34 +/- 0.52 nM (n = 3) at 4 degrees C and there were 3.43 +/- 0.78 (n = 3) pmoles of binding sites per mg of protein when homogenates of the whole epididymis were assayed. The concentration of ABP was lowest in the caput-corpus region of the epididymis, highest in the proximal cauda, and intermediate in the distal cauda. Essentially all of the ABP present in the distal cauda was intraluminal, as evidenced by the fact that flushing of the duct eliminated most of the [3H]5 alpha-DHT binding activity.     

Rationalizing translation attenuation in the network architecture of the unfolded protein response

Increased levels of unfolded proteins in the endoplasmic reticulum (ER) of all eukaryotes trigger the unfolded protein response (UPR). Lower eukaryotes solely use an ancient UPR mechanism, whereby they up-regulate ER-resident chaperones and other enzymatic activities to augment protein folding and enhance degradation of misfolded proteins. Metazoans have evolved an additional mechanism through which they attenuate translation of secretory pathway proteins by activating the ER protein kinase PERK. In mammalian professional secretory cells such as insulin-producing pancreatic β-cells, PERK is highly abundant and crucial for proper functioning of the secretory pathway. Through a modeling approach, we propose explanations for why a translation attenuation (TA) mechanism may be critical for β-cells, but is less important in nonsecretory cells and unnecessary in lower eukaryotes such as yeast. We compared the performance of a model UPR, both with and without a TA mechanism, by monitoring 2 variables: (i) the maximal increase in ER unfolded proteins during a response, and (ii) the accumulation of chaperones between 2 consecutive pulses of stress. We found that a TA mechanism is important for minimizing these 2 variables when the ER is repeatedly subjected to transient unfolded protein stresses and when it sustains a large flux of secretory pathway proteins which are both conditions encountered physiologically by pancreatic β-cells. Low expression of PERK in nonsecretory cells, and its absence in yeast, can be rationalized by lower trafficking of secretory proteins through their ERs.     

Cooperative interactions among afferents govern the induction of homosynaptic long-term depression in the hippocampus.

Prolonged periods of low-frequency stimulation have been shown to produce a robust, long-term synaptic depression (LTD) in both hippocampus and visual cortex. In the present study we have examined the extent to which interactions among afferents govern the induction of homosynaptic LTD in young-adult rats in hippocampal region CA1 in vitro. Field excitatory postsynaptic potentials were assessed before and after conditioning stimulation consisting of two 10-min trains of low-frequency stimulation (LFS; 1 Hz) of the Schaffer collateral/commissural pathway. LFS at an intensity producing a 0.5-mV response did not produce significant synaptic depression. However, LFS administered at a higher intensity resulted in significant input-specific LTD of a 0.5-mV test response. Picrotoxin, which also facilitates depolarization of CA1 neurons, significantly enhanced the magnitude of LTD after LFS at 0.5 mV. In addition, LFS at 0.5 mV in normal perfusion medium (no picrotoxin) produced only small changes in synaptic efficacy when either of two converging pathways was conditioned separately but produced a robust LTD when both pathways were conditioned simultaneously. This cooperative LTD was reversibly blocked by prior administration of 100 microM DL-aminophosphonovaleric acid but not by 20 microM nimodipine. Taken together, these results suggest that cooperative interactions among afferents contribute to voltage-dependent processes underlying the induction of homosynaptic LTD.     

Effect of prolactin, testosterone and estrogen on prolactin binding in the rat testis, prostate, seminal vesicle and liver

We have studied the hormonal control of prolactin (PRL) binding in the male rat sex glands and liver, subsequent to the recent demonstration and characterization of specific PRL binding sites in rat testis, prostate and seminal vesicle. Ovine PRL (200 micrograms/rat/day, 7 days) caused a time-dependent reduction in testicular binding of 125I-labelled PRL (measured 2 days after last injection) to 58% of control. Testosterone alone (1 mg/rat/day, 7 days) or PRL caused similar reductions in binding, while their coadministration further lowered PRL binding to 10% of control. The synergism of PRL and testosterone suggests that either these doses are submaximal, or that they are acting on different systems. Estradiol was administered as a single dose of 2 mg/rat and the PRL binding determined on day 10 and day 19 was reduced to 37% of control, as after testosterone. Addition of PRL whether from day 1 to day 7 or from day 11 to day 17 of estradiol injection had no effect, suggesting that the EB site of action is closer to the PRL receptor than that for PRL or testosterone. Estradiol resulted in a 72% reduction of PRL binding in the prostate, after 10 days, which subsequent PRL completely restored. PRL also partially restored the estradiol-induced time-dependent weight reduction of the prostate, but PRL coadministered from day 1 of estradiol did not inhibit the estradiol effects, suggesting a competitive mechanism for the two. While testosterone more than doubled PRL binding in the seminal vesicle, estradiol reduced it by 32% and organ weight by 21%. PRL given after estradiol restored the weight loss, but not the binding, suggesting that two different mechanisms of action are involved. In the liver, coadministration of testosterone with PRL could not inhibit the induction by PRL of its own hepatic sites, in keeping with a more direct site of action for PRL than for testosterone. These results demonstrate the profound effects of PRL, and of the sex steroids testosterone and estrogen, on PRL binding in the male sex glands and liver. The physiological implication of these findings on the role of PRL in male sexual function is currently being investigated.     

Neuronal circuitry regulates the response of Caenorhabditis elegans to misfolded proteins

The consequence of chronic protein misfolding is the basis of many human diseases. To combat the deleterious effects of accumulated protein damage, all cells possess robust quality-control systems, specifically molecular chaperones and clearance machineries, that sense and respond to protein misfolding. However, for many protein conformational diseases, it is unclear why this quality-control system does not efficiently counter protein aggregation. Previous findings that the heat shock response in Caenorhabditis elegans is regulated by thermosensory neurons led us to consider whether neuronal activity could also be responsible for the inadequate response of an organism to chronic protein misfolding. Here we show, in animals expressing polyglutamine expansion proteins and mutant SOD-1(G93A) in intestinal or muscle cells, that the nervous system does indeed control the cellular response to misfolded proteins. Whereas polyglutamine expansion-expressing animals with WT thermosensory neurons readily express protein aggregates, leading to cellular dysfunction without concomitant up-regulation of molecular chaperones, modulation of the nervous system results in chaperone up-regulation that suppresses aggregation and toxicity. The neuronal signal is transmitted through calcium-activated dense core vesicle neurosecretion. Cell-nonautonomous control of chaperone expression by the thermosensory neurons allows C. elegans to respond differently to acute stress such as heat shock, and chronic stress caused by the expression of misfolded proteins, suggesting that neuronal signaling determines the course of cellular proteotoxicity.     

疥螨虫体蛋白的小鼠免疫效应分析

目的观察疥螨全虫蛋白诱导BALB／c小鼠产生体液及细胞免疫应答水平。方法疥螨全虫蛋白皮下注射免疫BALB／c小鼠（100μg／只）,共疫3次,每次间隔2周,ELISA法检测小鼠的抗体水平、MTT比色法检测脾淋巴细胞增殖能力,及脾淋巴细胞培养上清分泌细胞因子水平。结果疥螨虫体蛋白能诱导小鼠产生特异性各类抗体（IgG、IgG1、IgG2a、IgE、IgM）;免疫小鼠的脾淋巴细胞增殖能力及脾细胞培养上清分泌4种细胞因子的水平（IL-2、IFN-γ、IL-4、IL-5）较PBS组、FCA组高,且差异有统计学意义（P〈0．05）。结论疥螨全虫蛋白能诱导小鼠产生较高水平的体液和细胞免疫应答,但对于其是否具有抵抗疥螨病的能力仍有待进一步研究。     

泌尿生殖道大肠埃希菌感染男性不育患者精浆NO、MDA和SOD含量的变化

目的探讨泌尿生殖道大肠埃希菌感染的男性不育患者精浆一氧化氮(NO)、丙二醛(MDA)和过氧化物歧化酶(SOD)水平变化对生精细胞的影响。方法60例泌尿生殖道大肠埃希菌感染男性不育患者(A组)、30例无感染不育患者(B组)和有正常生育能力的健康男性30例(C组),采用硝酸还原酶法测定精浆NO的含量,硫代巴比妥酸比色法测定精浆MDA含量,黄嘌呤氧化酶法测定精浆SOD的活力。选择大肠埃希菌感染不育组中精浆MDA含量17μmol/L 30例,精浆MDA的含量在7~9μmol/L之间的正常生育的健康男性20例,测定精子的各项运动参数并进行比较。结果A、B、C组精浆中NO含量分别为(137.64±25.87)(、117.17±27.72)和(66.88±20.51)μmol/L,MDA含量分别为(18.44±4.06)(、10.50±2.33)和(7.62±1.52)nmol/ml,SOD活力分别为(93.93±18.93)、(126.71±25.63)和(195.66.±19.26)μmol/L,A组与C组及B组与C组比较差异均有统计学意义(P均0.01)。MDA含量升高主要影响精子直线速度、前向性运动率,其次为平均移动角度、侧摆幅度、直线性百分率。经相关分析,感染性不育患者精浆MDA的含量与NO含量呈正相关(r=0.63),与SOD活力呈负相关(r=-0.69)。结论泌尿生殖道大肠埃希菌感染男性不育患者精浆NO、MDA含量及SOD活力异常可能是导致男性不育的原因之一。     

A study of the cellular and humoral immune response in patients with myelofibrosis

There was evidence of impaired cellular immunity in 10 patients with myelofibrosis. In-vitro lymphocyte transformation with phytohaemagglutin, concanavalin A, and dinitrochlorobenzene skin reaction were diminished. Signs of impaired humoral activity were also found, the primary response to a-Helix pomatia haemocyanin being impaired, particularly in the immunoglobulin-A class. Moreover three patients had a benign paraproteinaemia. Immune-complexes (1C) could be demonstrated with various test systems. The indirect granulocyte phagocytosis test was positive in 50%, the Clq-binding in 70% and the polyethylene glycol precipitation test in 50%. In most patients complement levels were normal, although the patient with the most advanced disease had low C3A, C3 and C4 levels accompanied by high levels of IC. No correlation could be shown between impaired immune response or levels of IC when they were related to spleen diameter or degree of anaemia. Some relation however existed between disturbed immune response and IC when they were related to time elapsed since diagnosis. It is suggested that the impaired immune response is the result of primary bone marrow disease and that the presence of IC may reflect the extent of fibrosis.