In Situ Photo-Crosslinkable Protein Bioadhesive for Bone Graft Fixation.

Bone grafting is a fundamental dental surgery procedure widely used for implant placement and periodontal disease management treatments. Despite its broad applications, vertical bone augmentation presents unique challenges, including the risk of graft displacement due to gravitational and masticatory forces. Traditional physical stabilization methods introduce additional complexities and risks, underscoring the need for innovative fixation technologies. This study aimed to develop an in situ photo-crosslinkable bioadhesive hydrogel (iPBAH) as a multifunctional bone graft binder to enhance the process of bone reconstruction. The bioadhesive is composed of mussel-derived adhesive protein (MAP) fused with the cell-adhesive peptide RGD. The numerous tyrosine residues in MAP facilitate rapid photo-crosslinking, enabling efficient hydrogel formation using visible blue light. Subsequently, iPBAH underwent comprehensive characterization to evaluate its suitability as a multifunctional bone graft binder. iPBAH efficiently underwent in situ crosslinking through harmless exposure to visible light within minutes and displayed several exceptional properties, including a microporous structure, underwater adhesion, extended durability, high compressive strength, and biocompatibility. In vivo assessments, using male Sprague-Dawley rats, demonstrated that iPBAH binder significantly enhanced bone regeneration in a rat calvarial bone defect model. The in situ crosslinking of the iPBAH binder during bone graft transplantation can effectively fill irregular and complex defect shapes while simultaneously preventing graft material leakage. The improved physical attributes of the bound graft material can enhance its resistance to external forces, thereby ensuring sustained retention over time. Moreover, the interaction between iPBAH and surrounding tissues promotes adhesion and integration of the graft material with host tissues in the defect area. In addition, the included RGD peptide in iPBAH can augment inherent cell recruitment, adhesion, and growth, consequently expediting osteogenesis.

Optical clearing agent reduces scattering of light by the stratum corneum and modulates the physical properties of coenocytes via hydration.

BACKGROUND: The interaction between light and the skin determine how the skin looks to the human eye. Light can be absorbed, scattered, and reflected by different components of the skin in a variety of different ways. Here, we focus on the scattering properties of the outmost layer, the stratum corneum (SC). However, we currently have limited methods with which to distinguish the scattering of light by SC from the changes due to other components of the skin. MATERIALS AND METHODS: Dark-field images of tape-striped corneocytes were used in vitro to study the differences in light scattered by the SC and other skin components. Several optical clearing agents (OCAs) were tested for their ability to reduce light scattering. Physical properties of the SC (water content, keratin configuration, and volume) after OCA treatment were investigated using FT-IR, confocal Raman microscopy, and 3D laser microscopy. RESULTS: Urea derivatives, several reducing sugars, and sugar alcohols, which were used as OCA in optics and also used as humectants in cosmetic area, could reduce scattering. However, unlike dehydration in optics, penetration of water into the keratin was increased at low OCA concentrations. In such conditions, the volume of corneocytes was increased but their stiffness was reduced. CONCLUSION: By analyzing the tape-striped SC, we were able to measure the changes in the optical and physical properties of corneocytes in response to OCAs. Hydration of the SC layer by OCAs reduces light scattering from the corneocytes and would be helpful in moisturizing the skin and helping the skin look healthy.

Staphylococcus aureus-derived membrane vesicles exacerbate skin inflammation in atopic dermatitis.

BACKGROUND: Skin colonization or infection with Staphylococcus aureus is known to trigger aggravation of atopic dermatitis (AD). However, the exact mechanisms by which S. aureus can worsen AD are unknown. OBJECTIVE: We investigated whether and how S. aureus-derived membrane vesicles (MVs) contribute to worsening of AD. METHODS: Immunohistochemical and immunoelectron microscopic analyses were performed to detect staphylococcal protein A (SPA) in the epidermis of AD lesions. HaCaT cells were treated with S. aureus MVs and were analysed for the expression of cytokine genes. Immunopathology and cytokine gene profiles were analysed after topical application of S. aureus MVs to AD-like skin lesions in a mouse model. RESULTS: The MV component SPA was detected in the keratinocytes as well as in the intercellular space of the epidermis of AD lesions colonized with S. aureus. Intact MVs from S. aureus delivered their components to keratinocytes and stimulated pro-inflammatory cytokine gene expression in vitro. A knock-down of Toll-like receptor 2 or nucleotide-binding oligomerization domain 2 using small interfering RNAs suppressed interleukin-8 gene expression. Topical application of intact S. aureus MVs to AD-like skin lesions in the mouse model induced massive infiltration of inflammatory cells and the resulting eczematous dermatitis. This inflammatory reaction was associated with a mixed Th1/Th2 immune response and enhanced expression of chemokine genes in AD-like skin lesions. CONCLUSIONS AND CLINICAL RELEVANCE: This study showed the importance of S. aureus MVs as a potent mediator for worsening of AD among many exogenous worsening factors of AD. Thus, S. aureus MVs may be regarded as one of the therapeutic targets for the management of AD aggravation.

The effect of sandblasting and different primers on shear bond strength between yttria-tetragonal zirconia polycrystal ceramic and a self-adhesive resin cement.

PURPOSE: To evaluate the effect of zirconia primers, air-abrasion, and tribochemical surface treatment methods on the shear bond strength between yttria-tetragonal zirconia polycrystal (Y-TZP) ceramic and self-adhesive resin cement. METHODS AND MATERIALS: Y-TZP ceramic surfaces were ground flat with 600-grit silicon carbide paper and then divided into seven groups of 10 and treated as follows: untreated (control), Monobond Plus, Z-PRIME Plus, ESPE Sil with CoJet, air-abrasion, Monobond Plus with air-abrasion, and Z-PRIME Plus with air-abrasion. Self-adhesive resin cement was placed onto the treated Y-TZP specimens for each group. All specimens were thermocycled and subjected to a shear bond strength test. Scanning electron microscope images of the fractured areas and x-ray diffraction (XRD) analysis of the surface-treated Y-TZP specimens were performed. Data were statistically analyzed using one-way analysis of variance and the Student-Newman-Keuls multiple comparison test (p<0.05). RESULTS: The Z-PRIME Plus treatment in combination with air-abrasion produced the highest bond strength (16.50±2.26 MPa), followed by air-abrasion (10.56±3.32 MPa), and then Monobond Plus combined with air-abrasion (8.93±3.13 MPa), ESPE Sil after CoJet application (8.54±3.98 MPa), and the Z-PRIME Plus group (8.27±2.79 MPa). The control (3.91±0.72 MPa) and Monobond Plus (4.86±1.77 MPa) groups indicated the lowest results (p<0.05). The XRD results showed the peaks of the monoclinic phase for the air-abrasion and CoJet treatment groups compared with the Y-TZP control. CONCLUSION: Z-PRIME Plus primer application after air-abrasion presented the best results for improving the bond strength between Y-TZP ceramic and self-adhesive resin cement.

Anatomical differences in lower third molars visualized by 2D and 3D X-ray imaging: clinical outcomes after extraction.

The purpose of this study was to evaluate the relationship between third molars and the inferior alveolar canal using panoramic radiographs and cone beam computed tomography (CBCT) scans and to assess clinical outcomes after third molar removal retrospectively. The degree of superimposition, buccolingual position (buccal, central, and lingual) and physical relationship (separation, contact, and involved) were measured using CBCT scanning. Post-extraction complications were recorded. Based on radiographic evaluation, 45.9% of third molar roots were inside the inferior alveolar canal, 21.3% were in contact with the inferior alveolar canal, and 32.8% were separated from the canal. The frequency at which the mandibular canal was separated from the root apex was significantly higher when the canal was in the buccal position (80.0%) than in the central (20.0%) and lingual positions (0.0%). Although on panoramic radiographs all third molars were directly superimposed on the inferior alveolar canal, CBCT showed direct contact or canal involvement in 67.2% and separation of the canal from the root apex in 32.8%. Complications occurred in nine patients: eight had third molar root apices inside or in contact with the inferior alveolar canal. The prevalence of post-extraction complications correlated with the absence of cortication around the inferior alveolar canal.

Natural orifice specimen extraction versus conventional laparoscopically assisted right hemicolectomy.

BACKGROUND: This case-control study compared the clinical outcomes of totally laparoscopic hemicolectomy with natural orifice specimen extraction (NOSE) and the conventional laparoscopically assisted approach for right-sided colonic cancer. METHODS: Consecutive patients who underwent totally laparoscopic mobilization of the right colon with transvaginal resection, anastomosis and specimen extraction between April 2007 and December 2009 were matched by various clinicopathological characteristics with patients who had conventional laparoscopically assisted procedures. RESULTS: Thirty-four patients in each group were studied. The number of lymph nodes harvested and the resection margin status were similar in the two groups. After NOSE, patients experienced less pain (mean(s.e.m.) pain score on day 1: 4·2(0·3) versus 5·7(0·3), P = 0·001; on day 3: 2·6(0·2) versus 3·5(0·2), P = 0·010) and had a shorter hospital stay (mean(s.d.) 7·9(0·8) versus 8·8(1·5) days; P = 0·003). The NOSE group had less surgical morbidity than the laparoscopically assisted group, but the difference was not significant (4 of 34 versus 9 of 34; P = 0·119). After a median follow-up of 23 (range 5-40) months, there was no transvaginal access-site recurrence or posterior colpotomy-related complications. NOSE was associated with significantly better cosmetic results (mean(s.d.) score 7·5(1·7) versus 6·6(1·8); P = 0·037). CONCLUSION: The NOSE approach is feasible with favourable short-term surgical outcomes.

Three-dimensional spin configuration of ferromagnetic nanocubes.

We have systematically investigated three-dimensional spin configurations in ferromagnetic nanocubes using micromagnetic simulation with variation of cube geometry. For thin cuboids, a spin configuration exhibits a four-domain Landau state with a magnetic vortex structure at the center as in the case of a thin film square. For a thick cube, a complex spin configuration with an S-type cylindrically asymmetric vortex having two cores on a pair of surfaces while a leaf-like and a C-type states are observed on the other two pairs of cube surfaces. Competition between the geometrical symmetry and magnetic energy minimization condition in ferromagnetic nanocubes leads to the complex spin structure with a spontaneously broken symmetry.

IVS10+12A>G polymorphism in hMSH2 gene associated with prognosis for patients with colorectal cancer.

BACKGROUND: The polymorphisms in DNA repair genes may contribute to a variation in the DNA repair capacity, thereby affecting the risk of carcinogenesis and prognosis of colorectal cancer. Accordingly, the present study analyzed 14 polymorphisms in DNA repair genes and their impact on the prognosis for patients with colorectal cancer. MATERIALS AND METHODS: Three hundred and ninety-seven consecutive patients with curatively resected colorectal adenocarcinoma were enrolled in the present study. The genomic DNA was extracted from fresh colorectal tissue and 14 polymorphisms of DNA repair genes determined using a real-time PCR genotyping assay. RESULTS: The median age of the patients was 63 years, and 218 (54.9%) patients had colon cancer, while 179 (45.1%) patients had rectal cancer. A multivariate survival analysis, including age, differentiation, carcinoembryonic antigen level, and stage, revealed a better survival for the patients with the combined IVS10+12AG and GG genotype than for the patients with the IVS10+12AA genotype [disease-free survival: hazard ratio (HR) 0.47, 95% confidence interval (CI) 0.30-0.75, P = 0.002; overall survival: HR 0.50, 95% CI 0.26-0.98, P = 0.042]. None of the other polymorphisms was associated with survival. CONCLUSION: The IVS10+12A>G polymorphism in the hMSH2 gene was found to be an independent prognostic marker for patients with colorectal cancer.

Uterine leiomyomas and their effect on in vitro fertilization outcome: a retrospective study.

PURPOSE: The effect of uterine leiomyomas on the outcome of in vitro fertilization (IVF) treatment has been controversial. This study was undertaken to clarify influence of fibroids on IVF success, in a large population with age and other potential confounding variables controlled for in the analysis. METHODS: A population of 141 patients with and 406 without leiomyomata undergoing their first IVF cycle was studied. RESULTS: The association between uterine leiomyomas and assisted reproduction treatment outcome was not statistically significant (OR = 0.73, 95% CI: 0.49-1.19, p = 0.21) after controlling for age and other risk factors. Also, fibroids neither affected the risk of spontaneous abortion (OR = 1.06, 95% CI: 0.44-2.60) nor the risk of ectopic pregnancy (OR = 0.78, 95% CI: 0.08-8.02). Location of fibroids (intramural vs. submucosal/subserosal) and their size had no significant effect on pregnancy outcome. CONCLUSIONS: Results from our analyses indicated that in vitro fertilization outcome was not affected by the presence of uterine leiomyomas. Therefore, in patients with normal uterine cavities and fibroids less than a certain size (i.e., < 7 cm), undergoing myomectomies as a prerequisite for assisted reproduction treatment is seriously questionable.

Microsatellite alteration in histologically normal lung tissue of patients with non-small cell lung cancer.

Microsatellite alteration (MSA) has been observed in a fraction of non-small cell lung cancer (NSCLC). Most prior studies regarding MSA in lung cancer have usually used adjacent non-malignant lung tissues as a source of constitutional DNA. However, these normal tissues might have genetic alterations because the entire field of bronchial tree is exposed to the same carcinogenic insult. The aim of this study was to search if MSA is present in the histologically normal lung tissue of patients with NSCLC. Tumor and corresponding normal lung tissue specimens were obtained from 20 patients with NSCLC. Normal lung tissue specimens were obtained from either the opposite end of resected surgical samples or as distant from the tumor as possible. They were examined histopathologically and confirmed as normal by H-E stain. Patients' peripheral lymphocytes were used as the source for the normal DNA. Sixteen markers on 3p and 9p (nine and seven markers, respectively) were used. MSA was detected in seven of 20 (35%) histologically normal lung tissue specimens at a frequency similar to that observed in tumor tissue (eight of 20, 40%). Five cases showed MSA in both normal lung tissue and the corresponding tumor. In these five cases, MSA in normal lung tissue was detected at the same microsatellite markers which MSA was detected in the corresponding tumor. The number and size of novel bands in normal lung tissue was identical to that in tumor tissue except in one case. In which case, the same pattern of MSA was found in both normal lung tissue and corresponding tumor tissue at two markers. However, at one marker, while one identical novel band was detected in normal lung tissue and corresponding tumor tissue, another novel band was found only in tumor tissue. In two of 12 patients whose tumor was negative for the presence of MSA, MSA was detected in normal lung tissue. These results indicate that genetic alterations are widely distributed in the lung tissue of patients with lung cancer and provide considerable support for the field cancerization theory. Screening for MSA in resected normal lung tissue might be a new method to identify patients at high risk for developing second primary lung cancers.

T-DNA insertional mutagenesis for functional genomics in rice.

We have produced 22 090 primary transgenic rice plants that carry a T-DNA insertion, which has resulted in 18 358 fertile lines. Genomic DNA gel-blot and PCR analyses have shown that approximately 65% of the population contains more than one copy of the inserted T-DNA. Hygromycin resistance tests revealed that transgenic plants contain an average of 1.4 loci of T-DNA inserts. Therefore, it can be estimated that approximately 25 700 taggings have been generated. The binary vector used in the insertion contained the promoterless beta-glucuronidase (GUS) reporter gene with an intron and multiple splicing donors and acceptors immediately next to the right border. Therefore, this gene trap vector is able to detect a gene fusion between GUS and an endogenous gene, which is tagged by T-DNA. Histochemical GUS assays were carried out in the leaves and roots from 5353 lines, mature flowers from 7026 lines, and developing seeds from 1948 lines. The data revealed that 1.6-2.1% of tested organs were GUS-positive in the tested organs, and that their GUS expression patterns were organ- or tissue-specific or ubiquitous in all parts of the plant. The large population of T-DNA-tagged lines will be useful for identifying insertional mutants in various genes and for discovering new genes in rice.

Tissue-preferential expression of a rice alpha-tubulin gene, OsTubA1, mediated by the first intron.

The genomic clone encoding an alpha-tubulin, OsTubA1, has been isolated from rice (Oryza sativa L.). The gene consists of four exons and three introns. RNA-blot analysis showed that the gene is strongly expressed in actively dividing tissues, including root tips, young leaves, and young flowers. Analysis of chimeric fusions between OsTubA1 and beta-glucuronidase (GUS) revealed that the intron 1 was required for high-level GUS expression in actively dividing tissues, corresponding with normal expression pattern of OsTubA1. Fusion constructs lacking the intron 1 showed more GUS staining in mature tissues rather than young tissues. When the intron 1 was placed at the distal region from 5'-upstream region or at the 3'-untranslated region, no enhancement of GUS expression was observed. Sequential deletions of the OsTubA1 intron 1 brought about a gradual reduction of GUS activity in calli. These results suggest that tissue-preferential expression of the OsTubA1 gene is mediated by the intron 1 and that it may be involved in a mechanism for an efficient RNA splicing that is position dependent.

Clinical characteristics of Peutz-Jeghers syndrome in Korean polyposis patients.

Peutz-Jeghers syndrome is an autosomal dominant inherited disorder characterized by hamartomatous polyps in the small bowel and mucocutaneous pigmentation. Patients with Peutz-Jeghers syndrome often present as surgical emergencies with complications of the polyps, such as intussusception, bowel obstruction, and bleeding. Recently an increased risk of malignancies has also been reported. This study was initiated to determine the clinical features of Peutz-Jeghers syndrome in Korean patients, with special attention to the development of malignancies. Thirty patients with Peutz-Jeghers syndrome were investigated; their median age was 23.5 years, and symptoms appeared at a median age of 12.5 years. Family history was positive in one-half of cases, and mucocutaneous pigmentation was observed in almost all patients (93%). The jejunoileum was the most frequent site of the polyps, and there were generally 10-100 polyps. Multiple laparotomies were performed in a substantial portion of the patients, due mainly to polyp-induced bowel obstruction, and the surgical interventions were begun at a relatively young age (average 21.4 years). Four cases of small-bowel cancer and one case of breast cancer were detected in probands, at a relatively young age (mean 36 years). Cancers of the small bowel, stomach, colon, breast and cervix were diagnosed in the first relatives of the probands. Close follow-up from an early age should thus be performed in patients with Peutz-Jeghers syndrome as they are at high risk of surgical emergency and development of malignancy.

Analysis of p73 in human borderline and invasive ovarian tumor.

p73 is a novel gene that has high sequence homology and similar gene structure to the tumor suppressor gene p53. We analysed p73 in seven ovarian carcinoma cell lines and a total of 63 human borderline and invasive ovarian tumor samples. Loss of heterozygosity at this locus was observed in 50% of invasive tumors but in none of the borderline tumors. Biallelic expression of the gene was observed in the heterozygous tumor tissues. Direct sequencing and single-strand conformation polymorphism analyses of the p73 cDNA sequence homologous to the highly mutatable region of p53 did not reveal any mutations. When compared to the primary cultures of normal human ovarian surface epithelial cells and immortalized cell lines, four of the seven ovarian carcinoma cell lines, 71% of the invasive tumors, and 92% of the borderline tumor tissues express elevated levels of p73 transcript. Except for the OVCA3 cell line, Western blot analysis of the nuclear extracts prepared from the cell lines showed concordant levels of p73 protein. Our analysis also demonstrated the expression of a spliced variant of p73 transcript with the omission of exon 2 solely in the cancer cell lines and invasive tumor tissues. This exon 2-spliced transcript would give rise to a truncated p73 protein without the N-terminal transactivation domain. In reminiscence of the dominant negative phenotype of the N-terminal truncated variants of another p53-related gene, p63, the expression of the truncated p73 variant form in ovarian tumors may play an important role in the pathogenesis of ovarian cancer.

A TGF-beta-inducible cell adhesion molecule, betaig-h3, is downregulated in melorheostosis and involved in osteogenesis.

Melorheostosis is a rare bone disease characterized by linear hyperostosis and associated soft tissue abnormalities. The skin overlying the involved bone lesion is often tense, shiny, erythematous, and scleodermatous. In order to look for genes differentially expressed between the normal and involved skin, we cultured skin fibroblasts from the skin lesions of several afflicted patients, and identified differentially expressed genes by reverse dot-blot hybridization. We found that the genes human TGF-beta-induced gene product (betaig-h3), osteoblast-specific factor 2, osteonectin, fibronectin, and type I collagen were all downregulated in the affected skin fibroblasts, with betaig-h3 the most significantly affected. The expression of betaig-h3 was induced by TGF-beta in both affected and normal fibroblasts. In an effort to determine the mechanism of bone and skin abnormalities in melorheostosis, we made recombinant betaig-h3. Both immobilized and soluble recombinant betaig-h3 proteins with or without an RGD motif inhibited bone nodule formation of osteoblasts in vitro. Taken together, our results suggest that altered expression of several adhesion proteins may contribute to the development of hyperostosis and concomitant soft tissue abnormalities of melorheostosis, with betaig-h3 in particular playing an important role in osteogenesis.

Cloning and molecular analyses of a gibberellin 20-oxidase gene expressed specifically in developing seeds of watermelon.

To understand the biosynthesis and functional role of gibberellins (GAs) in developing seeds, we isolated Cv20ox, a cDNA clone from watermelon (Citrullus lanatus) that shows significant amino acid homology with GA 20-oxidases. The complementary DNA clone was expressed in Escherichia coli as a fusion protein, which oxidized GA(12) at C-20 to the C(19) compound GA(9), a precursor of bioactive GAs. RNA-blot analysis showed that the Cv20ox gene was expressed specifically in developing seeds. The gene was strongly expressed in the integument tissues, and it was also expressed weakly in inner seed tissues. In parthenocarpic fruits induced by 1-(2-chloro-4-pyridyl)-3-phenylurea treatment, the expression pattern of Cv20ox did not change, indicating that the GA 20-oxidase gene is expressed primarily in the maternal cells of developing seeds. The promoter of Cv20ox was isolated and fused to the beta-glucuronidase (GUS) gene. In a transient expression system, beta-glucuronidase staining was detectable only in the integument tissues of developing watermelon seeds.

Intracytoplasmic injection of porcine, bovine, mouse, or human spermatozoon into porcine oocytes.

We determined the incidence of activation, male pronuclear formation, and apposition of pronuclei in porcine oocytes following intracytoplasmic injection of various porcine sperm components and foreign species spermatozoa, such as that of cattle, mouse or human. The porcine oocytes were activated by injection of a spermatozoon or an isolated sperm head. In contrast, injection of either sperm tail or a trypsin- or NaOH-treated sperm head failed to induce oocyte activation. Because injection of mouse, bovine, or human spermatozoon activated porcine oocytes, the sperm-borne activation factor(s) is not strictly species-specific. Male pronuclear formation and pronuclear apposition were observed in porcine oocytes following injection of porcine, bovine, mouse or human spermatozoa. Electrical stimulation following sperm cell injection did not enhance the incidence of male pronuclear formation or pronuclear apposition compared with sperm cell injection alone (P > 0.1). Following porcine sperm injection, the microtubular aster was organized from the neck of the spermatozoon, and filled the whole cytoplasm. In contrast, following injection of bovine, mouse, or human spermatozoon, the maternal-derived microtubules were organized from the cortex to the center of the oocytes, which seems to move both pronuclei to the center of oocytes. Cleavage to the two-cell stage was observed at 19-21 hr after injection of porcine spermatozoon. However, none of the oocytes following injection of mouse, bovine, or human spermatozoa developed to the mitotic metaphase or the two-cell stage. These results suggested that the oocyte activating factor(s) is present in the perinuclear material and that it is not species-specific for the porcine oocyte. Self-organized microtubules seemed to move the pronuclei into center of oocytes when foreign species spermatozoa were injected into porcine oocytes.

Anocutaneous advancement flap closure of high anal fistulas.

BACKGROUND: Fistula-in-ano may be treated by closing the internal fistulous opening. An anocutaneous flap was developed to facilitate this closure. METHODS: From October 1995 to April 1997, 40 patients with high trans-sphincteric or suprasphincteric anal fistulas were treated. The important components of the procedure are excision of the internal opening, excision or curettage of the tract, closure of the internal opening by an anocutaneous flap, and external drainage. RESULTS: In two patients (5 per cent) the flap separated and the sutured internal opening was exposed. One of the two was healed 4 weeks later. In the remaining 38 patients, complete healing occurred 2-3 weeks after operation. No patient was incontinent of gas or stool. CONCLUSION: This procedure is technically simple, heals rapidly with minimal scarring, and cures anal fistulas while preserving the anal sphincter.

Fertilization and in vitro development of porcine oocytes following intracytoplasmic injection of round spermatid or round spermatid nuclei.

The objective of this study was to determine fertilization rates and developmental ability of porcine oocytes following injection of round spermatid and round spermatid nucleus with artificial activation either 2 h before or immediately after injection. Electrical stimulation at 2 h before spermatid injection significantly increased the incidence of normal fertilization compared with that following injection without stimulation or with stimulation immediately after injection. Incidences of formation of 2 pronuclei and of apposition were not different in oocytes following intracytoplasmic spermatid and spermatid nucleus injection. Chromosome analysis revealed that most oocytes were diploid either following round spermatid or round spermatid nucleus injection. There was no diploid set of chromatin in oocytes at 20 h following sham injection. At 6 d following injection blastocoele formation was seen in the oocytes following round spermatid (25%) and round spermatid nucleus injection (27%). However, none of the oocytes developed to the blastocyst stage 6 d following sham injection. The average cell numbers of blastocysts 8 d after injection of spermatid and spermatid nucleus were 99 and 87, respectively. These results suggest that electrical stimulation before injection enhances the incidence of fertilization following round spermatid injection in the pig. Our study also indicates that either the round spermatid or it's nucleus can be used to produce viable embryos by injection into unfertilized porcine oocytes.

Fertilization of porcine oocytes following intracytoplasmic spermatozoon or isolated sperm head injection.

We demonstrated normal fertilization processes (as determined by pronuclear formation, pronuclear apposition and syngamy) in porcine oocytes either following intracytoplasmic spermatozoon (ICSI) or isolated sperm head injection. Microtubule organization and chromatin configuration were investigated in these oocytes during the first cell cycle. Following ICSI, the microtubular aster was organized from the neck of the spermatozoon and filled the whole cytoplasm. These male-derived microtubules appear to move both pronuclei to the center of oocytes. These cytoskeletal changes are analogous to those seen following conventional fertilization. In contrast, following isolated sperm head injection, the sperm aster was not seen. Instead, the microtubule matrix was organized from the cortex and then filled the whole cytoplasm in all cases in normally fertilized oocytes following injection (n=35). This organization is similar to what has been shown in the parthenogenetically activated oocytes. Chromosome analysis revealed that the oocytes injected with isolated sperm heads were fertilized normally. At 7 days following injection, the incidence of blastocoele formation following ICSI (38%) and isolated sperm head injection (22%) was higher than that following sham injection (2%). These results suggested that successful fertilization and preimplantation development occurred in porcine oocytes following either ICSI or isolated sperm head injection. Our results also indicated that fertilization processes can occur by self-assembled microtubules within cytoplasm in the absence of a sperm centrosome.