Increased salivary syndecan-1 level is associated with salivary gland function and inflammation in patients with Sjögren's syndrome.

OBJECTIVE: Syndecan-1 (SDC-1), a transmembrane heparin sulphate proteoglycan predominantly expressed on epithelial cells, also exists in a soluble form through ectodomain shedding. SDC-1 expression and shedding may be modulated in the inflammatory milieu of primary Sjögren's syndrome (SS). We investigated SDC-1 expression in minor salivary glands (MSGs) and analysed the association between salivary or plasma levels of SDC-1 and clinical parameters in SS. METHOD: We measured salivary and plasma SDC-1 levels via an enzyme-linked immunosorbent assay and assessed the salivary flow rates (SFRs) in 70 patients with SS and 35 healthy subjects. Disease activity indices, serological markers, salivary gland scintigraphy, and MSG biopsy were evaluated in patients with SS. RESULTS: SDC-1 expression was upregulated on ductal epithelial cells in inflamed salivary glands. Salivary SDC-1 levels in patients significantly exceeded those in healthy subjects [median (interquartile range) 49.0 (20.7-79.1) vs 3.7 (1.7-6.3) ng/mL, p < 0.001] and inversely correlated with SFRs (r = -0.358, p = 0.032) and ejection fractions of the parotid (r = -0.363, p = 0.027) and submandibular (r = -0.485, p = 0.002) glands in salivary gland scintigraphy. Plasma SDC-1 levels were significantly correlated with the EULAR Sjögren's Syndrome Disease Activity Index (r = 0.507, p < 0.001) and EULAR Sjögren's Syndrome Patient Reported Index (r = 0.267, p = 0.033). Focus scores were correlated with salivary SDC-1 levels (r = 0.551, p = 0.004). CONCLUSIONS: Salivary and plasma SDC-1 levels may constitute potential biomarkers for salivary gland function and disease activity, respectively, in SS.

Dicam promotes proliferation and maturation of chondrocyte through Indian hedgehog signaling in primary cilia.

OBJECTIVES: Primary cilium is required for mechano-biological signal transduction in chondrocytes, and its interaction with extracellular matrix is critical for cartilage homeostasis. However, the role of cilia-associated proteins that affect the function of cilia remains to be elucidated. Here, we show that Dicam has a novel function as a modulator of primary cilia-mediated Indian hedgehog (Ihh) signaling in chondrocytes. METHODS: Cartilage-specific Dicam transgenic mouse was constructed and the phenotype of growth plates at embryonic day 15.5 and 18.5 was analyzed. Primary chondrocytes and tibiae isolated from embryonic day 15.5 mice were used in vitro study. RESULTS: Dicam was mainly expressed in resting and proliferating chondrocytes of the growth plate and was increased by PTHrP and BMP2 in primary chondrocytes. Cartilage-specific Dicam gain-of-function demonstrated increased length of growth plate in long bones. Dicam enhanced both proliferation and maturation of growth plate chondrocytes in vivo and in vitro, and it was accompanied by enhanced Ihh and PTHrP signaling. Dicam was localized to primary cilia of chondrocytes, and increased the number of primary cilia and their assembly molecule, IFT88/Polaris as well. Dicam successfully rescued the knock-down phenotype of IFT88/Polaris and it was accompanied by increased number of cilia in tibia organ culture. CONCLUSION: These findings suggest that Dicam positively regulates primary cilia and Ihh signaling resulting in elongation of long bone.

Role of moesin in HMGB1-stimulated severe inflammatory responses.

Sepsis is a life-threatening condition that arises when the body's response to infection causes systemic inflammation. High-mobility group box 1 (HMGB1), as a late mediator of sepsis, enhances hyperpermeability, and it is therefore a therapeutic target. Despite extensive research into the underlying mechanisms of sepsis, the target molecules controlling vascular leakage remain largely unknown. Moesin is a cytoskeletal protein involved in cytoskeletal changes and paracellular gap formation. The objectives of this study were to determine the roles of moesin in HMGB1-mediated vascular hyperpermeability and inflammatory responses and to investigate the mechanisms of action underlying these responses. Using siRNA knockdown of moesin expression in primary human umbilical vein endothelial cells (HUVECs), moesin was found to be required in HMGB1-induced F-actin rearrangement, hyperpermeability, and inflammatory responses. The mechanisms involved in moesin phosphorylation were analysed by blocking the binding of the HMGB1 receptor (RAGE) and inhibiting the Rho and MAPK pathways. HMGB1-treated HUVECs exhibited an increase in Thr558 phosphorylation of moesin. Circulating levels of moesin were measured in patients admitted to the intensive care unit with sepsis, severe sepsis, and septic shock; these patients showed significantly higher levels of moesin than healthy controls, which was strongly correlated with disease severity. High blood moesin levels were also observed in cecal ligation and puncture (CLP)-induced sepsis in mice. Administration of blocking moesin antibodies attenuated CLP-induced septic death. Collectively, our findings demonstrate that the HMGB1-RAGE-moesin axis can elicit severe inflammatory responses, suggesting it to be a potential target for the development of diagnostics and therapeutics for sepsis.

CXC chemokine ligand 12a enhances chondrocyte proliferation and maturation during endochondral bone formation.

OBJECTIVE: We investigated the roles of CXC chemokine ligand 12a (CXCL12a), also known as stromal cell-derived factor-1α (SDF-1α), in endochondral bone growth, which can give us important clues to understand the role of CXCL12a in osteoarthritis (OA). METHODS: Primary chondrocytes and tibial explants from embryonic 15.5 day-old mice were cultured with recombinant mouse CXCL12a. To assess the role of CXCL12a in chondrogenic differentiation, we conducted mesenchymal cell micromass culture. RESULTS: In tibia organ cultures, CXCL12a increased total bone length in a dose-dependent manner through proportional effects on cartilage and bone. In accordance with increased length, CXCL12a increased the protein level of proliferation markers, such as cyclin D1 and proliferating cell nuclear antigen (PCNA), in primary chondrocytes as well as in tibia organ culture. In addition, CXCL12a increased the expression of Runx2, Col10 and MMP13 in primary chondrocytes and tibia organ culture system, implying a role of CXCL12a in chondrocyte maturation. Micromass cultures of limb-bud mesenchymal progenitor cells (MPCs) revealed that CXCL12a has a limited effect on early chondrogenesis, but significantly promoted maturation of chondrocytes. CXCL12a induced the phosphorylation of p38 and Erk1/2 MAP kinases and IκB. The increased expression of cyclin D1 by CXCL12a was significantly attenuated by inhibitors of MEK1 and NF-κB. On the other hand, p38 and Erk1/2 MAP kinase and NF-κB signaling were associated with CXCL12a-induced expression of Runx2 and MMP13, the marker of chondrocyte maturation. CONCLUSION: CXCL12a promoted the proliferation and maturation of chondrocytes, which strongly suggest that CXCL12a may have a negative effect on articular cartilage and contribute to OA progression.

Molecular survey of enteric viruses in commercial chicken farms in Korea with a history of enteritis.

Several enteric viruses have increasingly received attention as potential causative agents of runting-stunting syndrome (RSS) in chickens. A molecular survey was performed to determine the presence of a broad range of enteric viruses, namely chicken astrovirus (CAstV), avian nephritis virus (ANV), chicken parvovirus (ChPV), infectious bronchitis virus (IBV), avian rotavirus (AvRV), avian reovirus (ARV), and fowl adenovirus (FAdV), in intestinal samples derived from 34 commercial chicken flocks that experienced enteritis outbreaks between 2010 and 2012. Using techniques such as PCR and reverse-transcription PCR, enteric viruses were identified in a total of 85.3% of investigated commercial chicken flocks in Korea. Furthermore, diverse combinations of 2 or more enteric viruses were simultaneously identified in 51.7% of chicken farms positive for enteric viruses. The rank order of positivity for enteric viruses was as follows: ANV (44.1%), CAstV (38.2%), ChPV (26.5%), IBV (20.6%), ARV (8.8%), AvRV (5.9%), and FAdV (2.9%). Additionally, other pathogens such as Escherichia coli, Salmonella spp., Eimeria spp., and FAdV were detected in 79% of chicken flocks positive for enteric viruses using PCR, bacterial isolation, and microscopic examination. The results of our study indicate the presence of several enteric viruses with various combinations in commercial chicken farms that experienced enteritis outbreaks. Experimental studies are required to further understand the roles of enteric viruses in RSS in commercial chickens.

An outbreak of lymphomas in a layer chicken flock previously infected with fowlpox virus containing integrated reticuloendotheliosis virus.

Visceral lymphomas occurred in a 236-day-old layer flock previously diagnosed with reticuloendotheliosis virus (REV)-integrated fowlpox virus (FPV) infection at the age of 77 days. Common pathologic lesions were multiple neoplastic nodules of homogeneous lymphocytes in the livers and spleens of all submitted chickens. All neoplastic tissues were positive for the REV envelope (env) gene by PCR. In a retrospective molecular study of FPV-infected 77-day-old chickens from the same flock, we identified nearly full-length REV provirus integrated into the genome of FPV as well as the REV env gene in trachea samples, whereas only the REV LTR region was present in the FPV strain used to vaccinate this flock. The 622-bp REV env gene nucleotide sequence derived from the trachea and neoplastic tissues was identical. Commercial ELISA of serum samples revealed that all chickens aged between 17 and 263 days in this flock were positive for REV but not for avian leukosis virus. Taken together, the evidence suggests that the visceral lymphomas were caused by a REV-integrated FPV field strain. FPV infections of commercial chickens should be followed up by careful monitoring for manifestations of REV infection, including lymphomas and immune depression, considering the ease with which the REV provirus appears to be able to integrate into the FPV genome.

Artemisia fukudo essential oil attenuates LPS-induced inflammation by suppressing NF-kappaB and MAPK activation in RAW 264.7 macrophages.

In the present study, the chemical constituents of Artemisia fukudo essential oil (AFE) were investigated using GC-MS. The major constituents were alpha-thujone (48.28%), beta-thujone (12.69%), camphor (6.95%) and caryophyllene (6.01%). We also examined the effects of AFE on the production of nitric oxide (NO), prostaglandin E(2) (PGE(2)), tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6, in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Western blotting and RT-PCR tests indicated that AFE has potent dose-dependent inhibitory effects on pro-inflammatory cytokines and mediators. We investigated the mechanism by which AFE inhibits NO and PGE(2) by examining the level of nuclear factor-kappaB (NF-kappaB) activation within the mitogen-activated protein kinase (MAPK) pathway, which is an inflammation-induced signal pathway in RAW 264.7 cells. AFE inhibited LPS-induced ERK, JNK, and p38 phosphorylation. Furthermore, AFE inhibited the LPS-induced phosphorylation and degradation of Ikappa-B-alpha, which is required for the nuclear translocations of the p50 and p65 NF-kappaB subunits in RAW 264.7 cells. Our results suggest that AFE might exert an anti-inflammatory effect by inhibiting the expression of pro-inflammatory cytokines. Such an effect is mediated by a blocking of NF-kappaB activation which consequently inhibits the generation of inflammatory mediators in RAW264.7 cells. AFE may be useful for treating inflammatory diseases.

Development of compound microsatellite markers in red-tide-causing dinoflagellate Akashiwo sanguinea (Dinophyceae).

We isolated 13 polymorphic microsatellites from the red-tide causing dinoflagellate Akashiwo sanguinea. These loci were highly variable, with between 2 and 10 alleles per locus, and estimated gene diversity ranging from 0.08 to 0.82. These loci have the potential to reveal genetic structure and estimate gene flow among A. sanguinea populations.

Pseudomonas fluorescens HYK0210-SK09 offers species-specific biological control of winter algal blooms caused by freshwater diatom Stephanodiscus hantzschii.

AIMS: The study of an algicidal activity and mechanism of the isolated Pseudomonas fluorescens HYK0210-SK09 (SK09) against a winter bloomed harmful diatom, Stephanodiscus hantzschii. METHODS AND RESULTS: SK09 was isolated from the Paldang reservoir, Korea and used to biological control of S. hantzschii. The inoculation of SK09 at the final density of 5 x 10(6) cells ml(-1) caused degradation of >90% of S. hantzschii cells within 5 days. The algal cell lysis was achieved by a direct attack of the bacteria to the diatom cells, and the algicidal compound was located in the cytoplasm of the cell. As SK09 did not suppress Microcystis aeruginosa, Anabaena cylindrica, Coelastrum astroideum or Cyclotella meneghiniana, it appeared to attack S. hantzschii in a species-specific manner. Testing in an indoor mesocosms confirmed that SK09 effectively reduced S. hantzschii cells by 88% within 9 days. CONCLUSIONS: This bacterium is useful in regulating blooms of S. hantzschii. However, it should be studied in the future that their impact in shaping phytoplankton community and their activity in natural environments. SIGNIFICANCE AND IMPACT OF THE STUDY: The bacterium enabled us to develop a new strategy, to understand the interaction for anthropogenic control of harmful algal blooms in nature.

Enhancement of algicidal activity by immobilization of algicidal bacteria antagonistic to Stephanodiscus hantzschii (Bacillariophyceae).

AIMS: Enhancement of algicidal activity by immobilization of algicidal bacteria antagonistic to Stephanodiscus hantzschii. METHODS AND RESULTS: In laboratory studies, A diatom-lysing bacterium, Pseudomonas fluorescens HYK0210-SK09 showed strong algicidal activity against S. hantzschii, but a natural mesocosm study revealed that this bacterium failed to fully control natural blooms of Stephanodiscus at the low water temperatures that favour these blooms. Here, we sought to develop an effective immobilization strategy for enhancing the algicidal activity of HYK0210-SK09 in the natural setting. Bacterium HYK0210-SK09 was immobilized with various carriers including agar, alginate, polyurethane and cellulose sponge. The bacterial cells immobilized with cellulose sponge (CIS) induced more rapid and complete lysis of S. hantzschii than other carriers, and had a higher packing ability than polyurethane. Furthermore, CIS-immobilized cells showed higher lysis of S. hantzschii at the same concentrations as that of free cells (< or =1 x 10(7) cells ml(-1)), and had especially strong algicidal activity at the low temperatures (<10 degrees C). Based on these laboratory studies, we assessed the possible application of HYK0210-SK09 cells in the field by performing a mesocosm study during the winter season. The CIS-immobilized cells with species-specific activity towards the genera Stephanodiscus showed extremely high algicidal activity (up to 95%) against a bloom of Stephanodiscus hantzschii even at low water temperatures, because of high cell packing and subsequent cell protection against low temperatures and predators, whereas free cells showed negligible algicidal activities under these conditions. CONCLUSION: Immobilizing cells of HYK0210-SK09 in CIS foam, rather than in the other matrices tested, could achieve more efficient control of Stephanodiscus blooms and showed a significant algicidal activity on in vitro and in vivo blooms, even at low water temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: Collectively, these results indicate that CIS of algicidal bacteria may form an important strategy for effective management of Stephanodiscus blooms at low water temperatures.

Growth inhibition of bloom-forming cyanobacterium Microcystis aeruginosa by rice straw extract.

AIMS: To inhibit the growth of the bloom-forming cyanobacterium Microcystis aeruginosa using a rice straw extract. METHODS AND RESULTS: The cell numbers of the algal strain M. aeruginosa UTEX 2388 significantly decreased after treatment with different concentrations (0.01, 0.1, 1 and 10 mg l(-1)) of a rice straw extract for an 8-day cultivation period. Among seven tested allelochemicals from rice straw, salicylic acid at 0.1 mg l(1) exhibited the highest allelopathic activity (26%) on day 8. A synergistic effect on algal growth inhibition was found when adding two or three phenolic compounds from the rice straw. CONCLUSIONS: The growth of M. aeruginosa was inhibited by rice straw extract concentrations ranging from 0.01 to 10 mg l(1). This activity was due to the synergistic effects of various phenolic compounds in the rice straw. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of rice straw as an effective material for the growth inhibition of M. aeruginosa implies it may have the potential to be used as an environment-friendly biomaterial for controlling the algal bloom of M. aeruginosa in eutrophic water.

Protein C and/or S deficiency presenting as peripheral arterial insufficiency.

Although protein C and/or S deficiency has frequently been associated with venous thromboembolic events, instances of arterial thromboses have been reported. However, the exact incidence of protein C and/or S deficiency in patients with peripheral arterial insufficiency has not been established. Furthermore, given the lack of adequate studies to define the natural history and angiographic findings of these patients, the treatment has not been well delineated. Therefore, we conducted a prospective study to investigate the prevalence, characteristic angiographic findings and optimal treatments in patients with peripheral arterial insufficiency associated with protein C and/or S deficiency. Between September 2000 and August 2004, 133 patients who presented with peripheral arterial insufficiency underwent hypercoagulability tests before the initiation of any treatments. Of these, 11 patients (8.3%) with protein C and/or S deficiency were included in this study. There were nine males and two females. The ages ranged from 38 years to 72 years (mean 57 years). All patients showed characteristic angiographic findings: long segment thrombotic occlusion of a main peripheral artery without evidence of atherosclerosis or with mild atherosclerotic changes in the aorta and other major arterial trees. Surgical or endovascular procedures were performed in nine patients: bypass graft in four, thrombectomy in four and catheter-directed thrombolysis in one. Conservative treatment with full anticoagulation was performed in two patients. All patients received pre- and post-operative anticoagulation. Except for one amputated case, clinical and vascular laboratory improvements were achieved in 10 patients. Mean follow-up period was 21 months (range 4-45 months). However, one patient, in whom re-vascularization surgery was performed successfully, discontinued warfarin therapy himself at 10 months after surgery, graft occlusion and limb loss occurred at 30 months after surgery. This initial experience suggests that protein C and/or S deficiency may be an independent risk factor for peripheral arterial insufficiency. Patients who present with peripheral arterial insufficiency and protein C and/or S deficiency demonstrate characteristic angiographic findings. Once the diagnosis of protein C and/or S deficiency is made, patients should be treated with life-long anticoagulation.

Isolation and characterization of a bio-agent antagonistic to diatom, Stephanodiscus hantzschii.

AIMS: Identification of bacterium HYK0203-SK02 and its lysis of Stephanodiscus hantzschii. METHODS AND RESULTS: In an effort to identify a bio-agent capable of controlling S. hantzschii blooms, we used the algal lawn method to identify 76 bacteria in relevant water samples. Of these, the seven isolate showed algicidal activity against S. hantzschii; isolate HYK0203-SK02 exhibited the strongest algicidal activity, and was used for further analysis. 16S rDNA sequencing of this isolate allowed us to identify HYK0203-SK02 as a strain of Pseudomonas putida (99.2%). Growth of S. hantzschii was strongly suppressed by bacteria in all growth phases, with the strongest algicidal activity noted against diatoms in the exponential stage (5-18 days). Host range assays revealed that isolate HYK0203-SK02 also strongly inhibited the growth of Microcystis aeruginosa, but stimulated growth of the diatom Cyclotella sp., which has a similar structure to that of S. hantzschii. Biochemical assays revealed that the algicidal substance seemed to be localized in the cytoplasmic membrane of this newly identified algicidal bacterium. CONCLUSION: The algicidal bacteria P. putida HYK0203-SK02 caused cell lysis and death of not only diatom S. hantzschii but also cyanobacteria M. aeruginosa, dramatically. Algicidal substance might be located at the compartment of cytoplasmic membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: Taken together, our results indicate that P. putida HYK0203-SK02 may be a potential bio-agent for future use in controlling freshwater diatomic blooms.

Cytomegalovirus infection of the graft duodenum and urinary bladder after simultaneous pancreas-kidney transplantation.

Cytomegalovirus (CMV) is an important cause of morbidity after solid organ transplantation. We report a case of CMV infection involving the transplanted duodenum that developed after simultaneous pancreas-kidney transplantation. The patient, a 30-year-old woman with insulin-dependent diabetes undergoing hemodialysis due to chronic renal failure, received a simultaneous cadaveric pancreas-kidney transplantation. The exocrine secretion was diverted using bladder drainage. Immunosuppression was maintained by a combination of tacrolimus, mycophenolate mofetil, and steroids together with OKT3 induction. Both the donor and the recipient were serologically positive for CMV IgG CMV prophylaxis consisted of a short course of parenteral gancyclovir. The patient was discharged on postoperative day 39 with normal pancreas and kidney function. She presented 2 months after transplantation with hematuria. Cystoscopic pancreas allograft biopsy specimens showed evidence of tissue invasive CMV infection in the graft duodenum and bladder. The CMV antigenemia test was positive. At 4 months after transplantation, the patient underwent surgery with the diagnosis of acute abdomen. The surgical findings consisted of a diffuse acute purulent peritonitis due to perforation of the duodenal graft. We sutured the perforation with nonreabsorbable material. The CMV antigenemia test was negative. Eight days later, the patient developed massive hematuria. At surgery, the graft was removed. The patient was discharged from the hospital with normal renal function. Pathological study of the removed graft showed the duodenal segment to have multiple wide ulcers with CMV inclusions in epithelial cells.

The origin of cells that repopulate patellar tendons used for reconstructing anterior cruciate ligaments in man.

Cryopreserved patellar tendon allografts are often recommended for reconstruction of anterior cruciate ligaments (ACLs) because living donor fibroblasts are thought to promote repair. Animal studies, however, indicate that ligaments regenerate from recipient rather than donor cells. If applicable to man, these observations suggest that allograft cell viability is unimportant. We therefore used short tandem repeat analysis with polymerase chain reaction (PCR) amplification to determine the source of cells in nine human ACLs reconstructed with cryopreserved patellar tendon allografts. PCR amplification of donor and recipient DNA obtained before operation and DNA from the graft obtained two to ten months after transplantation revealed the genotype of cells and showed only recipient cells in the graft area. Rather than preserve the viability of donor cells, a technique is required which will facilitate the introduction of recipient cells into patellar tendon allografts.

Deep venous thrombosis associated with protein C and/or S deficiency: management with catheter-directed thrombolysis.

We performed this study to evaluate the efficacy of catheter-directed thrombolysis with urokinase in treating acute symptomatic iliofemoral deep venous thrombosis associated with protein C and/or S deficiency. A total of 42 consecutive patients with deep venous thrombosis were seen between September 2000 and August 2002. Of these, catheter-directed thrombolysis via the popliteal vein was performed in 5 patients (11.9%) with acute iliofemoral deep venous thrombosis associated with protein C and/or S deficiency. Average duration of symptoms was 4.2 days (range, 1-7 days). The average urokinase dose was 2.7 million IU (range, 0.6 million to 7.0 million IU) infused over an average of 33.1 h (range, 16-67 h). Lysis was complete in all five treated cases. Two cases had underlying iliac venous stenoses (>50%) that were treated with angioplasty and stent placement. In one patient in whom recanalization of a right iliac vein occlusion was successful, thrombosis occurred in the treated vein within 3 weeks of intervention despite full anticoagulation therapy, and further intervention was required. There were no complications or clinically detectable pulmonary emboli. The technical and clinical success rates were 100%. This initial experience suggests that catheter-directed thrombolysis for treatment of acute symptomatic iliofemoral deep venous thrombosis associated with protein C and/or S deficiency is safe and effective.

Effects of fish introduction on the length of the tail of cryptomonads in mesocosm experiments.

Two different morphs of the small cryptomonad, Plagioselmis prolonga var. nordica with a posterior tail, were observed during summer and fall in the hypertrophic lake, Lake Kasumigaura, Japan. The tail shortened in mesocosms stocked with planktivorous silver carp ( Hypophthalmichthys molitrix Val.) and elongated by more than 50% in mesocosms from which silver carp were removed. The density of Plagioselmis cells increased significantly upon fish stocking and decreased upon fish removal. The tail length was negatively correlated with algal abundance and positively correlated with crustacean densities, but there was no correlation with nutrient levels or physical environmental parameters in the mesocosms. The variation in tail length was induced by the presence/absence of fish, but was not related to their density. However, silver carp manipulation strongly affected the density of the majority of zooplankters and, interestingly, there was a strong correlation between zooplankton density and tail-length change in Plagioselmis. We propose a possible herbivore-induced defense mechanism triggered by the top predator, silver carp.

Development of rat oocytes following intracytoplasmic injection of sperm heads isolated from testicular and epididymal spermatozoa.

The possibility of obtaining normal development of rat oocytes following intracytoplasmic injection of rat sperm heads, obtained by sonicating spermatozoa from testes and epididymides, was evaluated. Irrespective of the source of spermatozoa, sperm heads were successfully injected into approximately 45% of oocytes used; after 9-12h of culture, approximately 55% of injected oocytes still had normal morphology. Of the oocytes injected with testicular sperm heads 45% were activated, with a female pronucleus and a second polar body, but significantly more oocytes (approximately 68%) injected with caput and cauda epididymal sperm heads were activated. Male pronuclear formation was observed in 67-84% of the activated oocytes, with no difference in the proportions among the different sources of sperm heads. When zygotes showing two pronuclei and a second polar body at 10h after injection were cultured in conditions that support development of 1-cell embryos produced in vivo, no embryos derived from testicular sperm heads developed to blastocysts after 120 h of culture. Development of embryos derived from cauda sperm heads was significantly higher at all points of assessment, while embryos from caput sperm showed an intermediate degree of development, compared with embryos from testicular spermatozoa. However, similar proportions (2-4%) of 1-cell embryos derived from all three groups of sperm heads developed into normal offspring after transfer to foster mothers; of the limited number of offspring tested, all were fertile. These results demonstrate that sperm heads from all sources tested are similar in their ability to contribute to full development of normal, fertile offspring.