Plumping fluid added to storage medium increases twofold the functional life span of fowl spermatozoa in vitro at 4°C.

1. The objective of this study was to examine whether addition of plumping fluid (PF) to Lake's solution (LS) for storage of fowl spermatozoa in vitro at 4°C can prolong survival and improve the quality of spermatozoa. 2. In experiment 1, aliquots of spermatozoa were stored in vitro in LS alone and LS containing 10%, 25%, 50% and 75% (v:v) PF for 0.5, 24, 48, 72, 96 and 120 h at 4°C. After the end of each storage period, spermatozoa were evaluated for their viability, mobility and penetrability. Viability was determined using SYBR-14 and propidium iodide (PI) staining. Mobility was assessed using an Accudenz assay. Penetrability was assessed using spermatozoa-inner perivitelline layer (IPL) interaction assay. 3. In experiment 2, aliquots of spermatozoa were stored in vitro in LS alone and LS containing 25% and 50% (v:v) PF for 0.5, 24, 48 and 72 h at 4°C, and then fertility of the spermatozoa was evaluated using intravaginal artificial insemination (AI) in hens. 4. Storage of spermatozoa in LS alone resulted in loss of viability, mobility, penetrability and fertility within 48 h. In contrast, no loss of viability and penetrability was observed for the spermatozoa stored for 48, 96, 72 and 48 h in LS containing 10%, 25%, 50% and 75% (v:v) PF, respectively. In particular, fertilising capacity was not lost for the spermatozoa stored in the presence of 25% or 50% PF in LS for 48 and 24 h, respectively. 5. In conclusion, these findings demonstrated that in vitro exposure of fowl spermatozoa to PF during hypothermic storage in LS prolonged spermatozoa survival. A 25% (v:v) level of inclusion of PF in LS may be effective for the improvement of viability, penetrability and fertilising ability of the stored spermatozoa.

Acrosome reaction of fowl sperm: evidence for shedding of the acrosomal cap in intact form to release acrosomal enzyme.

The aim of this study was to determine the site of enzyme release from the acrosome and the fate of the acrosomal cap during the process of acrosome reaction (AR) in fowl sperm. Gelatin substrate coverslips with halos were subjected to scanning electron microscopy to determine the site from which acrosomal proteolytic enzyme was released to form a halo around the acrosome of individual sperm. Aliquots of sperm treated with solubilized inner perivitelline layer (IPL) containing 5 mmol CaCl(2) were simultaneously subjected to fluorescent staining with fluorescein isothiocyanate-labeled peanut agglutinin and scanning electron microscopy to evaluate AR of sperm and to examine the status of the acrosomal region, respectively. Inside the halos, a gelatin-free (proteolyzed gelatin) layer was found extending some distance around the acrosome of sperm. All of the sperm showing the formation of halos on gelatin had a single circular opening around their subacrosomal rod at the base of the acrosomal cap. Interaction of sperm with solubilized IPL in the presence of 5 mmol CaCl(2) resulted in 41.4 ± 1.8% of the sperm to undergo AR, as evaluated by fluorescein isothiocyanate-labeled peanut agglutinin. Similarly, as observed using scanning electron microscopy, 38.2 ± 2.3% of the sperm treated with solubilized IPL plus 5 mmol CaCl(2) had exposed subacrosomal rod. In all sperm examined, no sign of disruption of the acrosomal membrane was found in the apical region of the acrosome. Rather, the acrosomal caps were found intact detached from the acrosomal region of the sperm, indicating that AR of fowl sperm resulted in the intact removal of the acrosomal cap. Based on these experimental observations, we suggest that the process of AR in fowl sperm is unique; the release of the acrosomal proteolytic enzyme may occur through a single circular opening formed at the base of the acrosomal cap and the acrosomal cap is detached in intact form from the posterior acrosomal region of the sperm.

Maturational changes in the survivability and fertility of fowl sperm during their passage through the male reproductive tract.

The objective of this study was to examine whether domestic fowl (Gallus domesticus) sperm undergo maturation in their capacity for survival and fertilization in the male reproductive tract. Sperm collected from the testis, epididymis and the proximal, middle and distal vas deferens were simultaneously stored in vitro in minimum essential medium (MEM) at 39°C for 0, 3 and 6h, and at 4°C for 24 and 48h. Sperm membrane integrity was measured using the dual fluorescent stain SYBR-14/propidium iodide (PI). Aliquots of sperm from the various sites were subjected to artificial insemination (AI) into the uteri of hens to assess the duration of sperm survival in the oviduct and to determine the fertility status of the sperm. Testicular sperm exhibited a very low capacity to survive under in vitro liquid storage conditions, irrespective of the storage temperature used, and in the oviduct, and they had a low ability to fertilize the ovum. On the contrary, sperm from the distal vas deferens had a higher survival rate during in vitro storage periods, a longer life span in the oviduct, and high fertility. Survival and fertilizing capacity of the sperm recovered from the testes increased gradually (P<0.05) from the testes to the distal vas deferens. In conclusion, we suggest that fowl sperm may undergo functional maturation through a process of gradual changes in their survival and fertilization capacities during their passage through the successive parts of the male reproductive tract.

Maturational changes in motility, acrosomal proteolytic activity, and penetrability of the inner perivitelline layer of fowl sperm, during their passage through the male genital tract.

The objective was to examine, in vitro, the motility, acrosomal proteolytic activity (APA), and penetrating ability of fowl sperm recovered from the testis and epididymis, as well as the proximal, middle, and distal vas deferens, to assess the potential fertilizing ability of sperm as a function of maturation. A motile sperm separation technique was used to estimate sperm motility with Accudenz, a gelatin slide technique was used to measure the diameter of the halo around the acrosome of individual sperm as an indication of APA, and a sperm-inner perivitelline layer (IPL) interaction assay was done to estimate the number of hole formations as an indication of sperm penetration into the IPL. Sperm in the testis exhibited the least motility, produced the smallest halos, and created the least number of holes per 0.25 mm(2). Motility, diameter of the halo, and number of holes increased gradually (P < 0.05) from the epididymis to the distal vas deferens and were markedly different (P < 0.05) between testicular and deferent duct sperm. Based on these in vitro experimental findings, we inferred that fowl sperm undergo a gradual process of maturational changes in motility, APA, and penetrability as a means of acquiring potential fertility during their passage throughout the male genital tract.

Dose response to vaginal administration of 1,25-dihydroxyvitamin D3 to cows.

It was previously reported that intravaginal (IVAG) administration of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) might be protective against bovine hypocalcaemia. In the present study, various doses of exogenous 1,25(OH)(2)D(3) were administered IVAG to ovariectomised cows, and the subsequent changes in the biochemical parameters of the blood were measured to assess the characteristics of vaginal absorption. Five cows received 1,25(OH)(2)D(3) IVAG at a dose of 0.125, 0.25, 0.5, or 1.0microg/kg of body weight (BW) or intravenously at a dose of 1.0microg/kg BW. Dosing was at intervals of at least two weeks in a 5x5 Latin square design. Vaginally administered 1,25(OH)(2)D(3) was absorbed in a dose-dependent manner. There was no correlation between the IVAG dose of 1,25(OH)(2)D(3) and subsequent changes in plasma calcium concentrations. The bioavailability of 1,25(OH)(2)D(3) administered IVAG at 1.0microg/kg BW was approximately 93%.

Striated microfilament bundles attaching to the plasma membrane of cytoplasmic bridges connecting spermatogenic cells in the black snail, Semisulcospira libertina (Mollusca, Mesogastropoda).

Striated microfilament bundles attaching to the plasma membrane of cytoplasmic bridges between spermatogenic cells are described in the black snail, Semisulcospira libertina. The bundles were occasionally observed in bridges connecting spermatogonia, spermatocytes and typical spermatids. Relations between bundles and centrioles could not be detected. The bundle had electron dense cross bands with a periodicity of approximately 200 nm, and attached to the membrane with almost right angle at the cross linker level. Phalloidin cytochemistry revealed that the bundle contained F-actin. In a case, a bundle connected two cytoplasmic bridges.

Technical note: Vaginal absorption of 1,25(OH)2D3 in cattle.

Although exogenous 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] administered via intravenous, intramuscular, and oral routes has been tested for efficacy in preventing parturient hypocalcemia in dairy cows, there are few reports concerning intravaginal administration. For this study, 1,25(OH)2D3 was administered via the bovine vaginal lumen, and subsequent changes in blood chemistry, including levels of 1,25(OH)2D3 and several minerals, were measured to confirm vaginal absorption. Each of 5 heifers received a single intravaginal dose of 1 microg of 1,25(OH)2D3/kg body weight; a single control heifer received the ethanol carrier alone. In heifers receiving 1,25(OH)2D3, the plasma 1,25(OH)2D3 levels increased markedly from baseline (88.3 +/- 20.3 pg/mL) within 2 h and reached a peak at 6 h after treatment (1967.4 +/- 1139.6 pg/mL). Plasma Ca levels increased from baseline (10.4 +/- 0.4 mg/dL) to a peak of 11.96 +/- 0.7 mg/dL at 24 h. The levels of inorganic phosphate in plasma increased over time from 7.3 +/- 0.5 to 8.1 +/- 0.8 mg/dL by 6 h and were maintained at a plateau level (9.1 +/- 0.7 to 8.6 +/- 0.6 mg/dL) from 24 to 96 h after treatment. Plasma magnesium decreased from a baseline level of 2.1 +/- 0.1 mg/dL to a plateau level of 1.8 +/- 0.1 mg/dL, which was sustained from 24 to 48 h after treatment. The present study provides evidence of the absorption of exogenous 1,25(OH)2D3 from the bovine vaginal wall, as shown by the marked elevation of plasma 1,25(OH)2D3 levels by 2 h after administration, and indicates the possible utility of intravaginal administration of 1,25(OH)2D3 for prophylaxis of hypocalcemia.

Long-term outcome after allogeneic bone marrow transplantation for hematological malignancies with non-remission status. Results of a single-center study of 24 patients.

To investigate the problem of allogeneic bone marrow transplantation (allo-BMT) for advanced stage patients, we retrospectively analyzed 24 consecutive patients who underwent allo-BMT in the non-remission stage. Twenty-four patients (19 males and 5 females) with acute leukemia, chronic myelogenous leukemia, and malignant lymphoma underwent allo-BMT. The patients had a median age of 30 years. There were eight cases of acute myelogenous leukemia (AML), six cases acute lymphocytic leukemia (ALL), nine cases of chronic myelogenous leukemia (CML), and one case of Burkitt's lymphoma. The 3-year overall survival rate was 22.5%, with a median survival time of 206 days in AML, 345 days in ALL, and 363 days in CML. Overall survival was associated with a recovery of platelets of less than 30 days and an acute graft-versus-host disease (acute GVHD) presence of less than grade II ( p=0.042). Fourteen patients died of transplantation-related diseases. Our important problem is to decrease transplantation-related deaths in allo-BMT during the non-remission stage, and longer survival can be expected with better pretreatment and prophylaxis for GVHD. In addition, the selection of the source of hematopoietic stem cell transplantation at an optimal time is considered to be another problem to be approached.

Combination therapy with irinotecan and cisplatin as neoadjuvant chemotherapy in locally advanced cervical cancer.

To evaluate the response rate and toxicity of the combination of irinotecan (CPT-11) and cisplatin in a neoadjuvant setting, a phase II study was conducted regarding the regimen of this combination in patients with locally advanced cervical cancer. Eligibility included patients with previously untreated stage Ib2, IIb, or IIIb squamous cell carcinoma with good performance status. CPT-11 (60 mg m(-2)) was administered intravenously on days 1, 8 and 15, followed by cisplatin (60 mg m(-2)) given intravenously on day 1. Treatment was repeated every 4 weeks for a total of two or three cycles. Among 23 eligible patients (median age: 59 years), three showed complete response (13%), 15 showed partial response (65%), for an overall response rate of 78% (95% confidence interval 58-90%). Stable disease was observed in four cases (17%) and progressive disease in one (4%). The median time to failure and median survival time have not yet been reached. Of the 52 treatment cycles administered, diarrhoea and grade 3 or 4 neutropenia were observed in 10% and 75% respectively. There were no therapy-related deaths. The combination of CPT-11 with cisplatin is a promising regimen for neoadjuvant chemotherapy in locally advanced cervical cancer. The toxicities of this regimen are well tolerated.

Solitary splenic recurrence of ovarian cancer: case report and review of the literature.

We report a rare case of solitary recurrence of ovarian cancer in the spleen which developed 4 years after initial treatment. Only six cases have been reported in the literature and all were serous carcinoma. Our patient had a splenectomy without any complications but developed a liver metastasis 10 months after splenectomy.

Two cases of endometrial adenocarcinoma arising from atypical polypoid adenomyoma.

Atypical polypoid adenomyoma (APA) most frequently presents as an endometrial polyp in premenopausal women and is believed to follow a benign course. In hysterectomy specimens from postmenopausal Japanease women, the endometrium contained an APA with an area of endometrial adenocarcinoma. A convincing transition zone between the APA and the adenocarcinoma was seen in our cases, suggesting that APA may develop into endometrial adenocarcinoma in postmenopausal women.

Irinotecan (CPT-11) combined with cisplatin in patients with refractory or recurrent ovarian cancer.

Irinotecan hydrochloride (CPT-11) is reportedly effective for the treatment of refractory or recurrent ovarian cancer. We investigated the antitumor efficacy and toxicity of combination therapy with CPT-11 and cisplatin in 25 patients (mean age 55 years, range 35-73 years) with refractory or recurrent ovarian cancer who had previously undergone platinum-based combination chemotherapy. Patients received two or more courses of treatment consisting of 50 or 60 mg/m2 of CPT-11 on days 1, 8 and 15 and 50 or 60 mg/m2 of cisplatin on day 1 administered intravenously. All patients were evaluable for the response and the toxicity profile. Complete responses were obtained in two (8.0%) patients and partial responses were obtained in eight (32.0%) patients, giving an overall response rate of 40% (10 of 25 patients) (95% CI 23.0-59.0%). The median duration of response was 5.5 months (range 2-27 months), the median time to tumor progression was 6 months (range 3-28 months) and the median overall survival was 12 months (range 3-39+ months). Grade 3 or 4 neutropenia, which was the most frequent and severe toxic effect, occurred in 36 (54.5%) of the 66 treatment courses and in 16 (64.0%) of 25 patients. The nadir of the leukocyte count occurred on days 18-19. Neutropenia was reversed by short-term administration of granulocyte colony-stimulating factor for 2-10 days. Less serious hematologic effects and non-hematologic effects, such as diarrhea, were also observed. This preliminary study showed that this regimen of CPT-11 and cisplatin was effective in patients with recurrent ovarian cancer.

Ovarian endometrioid adenocarcinoma coexistent with benign and borderline endometrioid adenofibroma: a case report.

A well-differentiated endometrioid adenocarcinoma coexistent with benign and borderline-malignant endometrioid adenofibroma was found in the ovary of a 64-year-old woman. She had vaginal bleeding caused by simple hyperplasia of the endometrium due to high levels of sex steroid hormones. A FIGO stage Ia solid ovarian tumor was identified. It was composed of irregularly shaped endometriotic glands with benign and borderline malignant cytologic features embedded in abundant fibromatous stroma. Well-differentiated malignant epithelium was adjacent to these areas, but fibromatous stroma was not predominant. She was treated by surgery and three cycles of chemotherapy. This paper describes this unusual tumor and reviews the literature.

A pregnant woman with clear cell adenocarcinoma of the ovary arising from endometriosis and with benign and borderline adenofibroma of the clear cell and endometrioid types.

We encountered a case of ovarian cancer in a 33-year-old, 8-week pregnant woman. Histological examination revealed both a transitive form of ovarian endometriosis with marked decidual changes due to pregnancy and clear cell carcinoma. Benign and borderline clear cell adenofibroma and benign and borderline endometrioid adenofibroma were also found. Parts of these adenofibromas showed transformation to clear cell carcinoma. This case suggests that clear cell carcinoma can arise from clear cell adenofibromas and/or ovarian endometriosis, even in young patients.

Yeast counterparts of subunits S5a and p58 (S3) of the human 26S proteasome are encoded by two multicopy suppressors of nin1-1.

Nin1p, a component of the 26S proteasome of Saccharomyces cerevisiae, is required for activation of Cdc28p kinase at the G1-S-phase and G2-M boundaries. By exploiting the temperature-sensitive phenotype of the nin1-1 mutant, we have screened for genes encoding proteins with related functions to Nin1p and have cloned and characterized two new multicopy suppressors, SUN1 and SUN2, of the nin1-1 mutation. SUN1 can suppress a null nin1 mutation, whereas SUN2, an essential gene, does not. Sun1p is a 268-amino acid protein which shows strong similarity to MBP1 of Arabidopsis thaliana, a homologue of the S5a subunit of the human 26S proteasome. Sun1p binds ubiquitin-lysozyme conjugates as do S5a and MBP1. Sun2p (523 amino acids) was found to be homologous to the p58 subunit of the human 26S proteasome. cDNA encoding the p58 component was cloned. Furthermore, expression of a derivative of p58 from which the N-terminal 150 amino acids had been removed restored the function of a null allele of SUN2. During glycerol density gradient centrifugation, both Sun1p and Sun2p comigrated with the known proteasome components. These results, as well as other structural and functional studies, indicate that both Sun1p and Sun2p are components of the regulatory module of the yeast 26S proteasome.

Cuticular plasticization induced by copulatory stimuli in female Haemaphysalis longicornis (Acari:Ixodidae).

Partially engorged adult female ticks, Haemaphysalis longicornis Neumann, received complete, incomplete, or no copulatory stimuli. Subsequently, they were inflated with physiological saline by injection into the haemocoel. Ticks that copulated received both preinseminational and inseminational stimuli and increased their body volume > 10-fold. Ticks that incompletely copulated received only preinseminational stimuli and expanded slightly, whereas ticks that did not copulate did not expand. These results show that the soft integument of H. longicornis only expanded after complete copulation. Therefore, copulatory stimuli induced cuticular plasticization.

Histological evaluation of skin reconstruction using artificial dermis.

An artificial dermis, composed of a collagen matrix, was applied to a full-thickness skin defect prepared on the back of rats. Two weeks later, a thin split-thickness skin autograft was overlaid on the matrix at each recipient site. The dermal layer at the recipient sites was 1.02 mm thick with prior application of artificial dermis, as compared with the 0.46 mm thickness observed without such pretreatment. Histologically, the split-thickness skin graft normally lies with no gap on the artificial dermis, which looks like natural dermis. Six days after grafting, the epithelial basal cells in the grafts showed an active uptake of bromodeoxyuridine (a thymidine analogue), indicating high activity of cell proliferation. About 50 and 20% respectively of the artificial dermis remained at each recipient site at 12 and 20 weeks after its application (after the skin defect). This finding indicates that bovine collagen, which is a constituent of the artificial dermis, is gradually replaced by the host tissue.

Nature and origin of cerebrovascular nerves with substance P immunoreactivity in bats (Mammalia: Microchiroptera), with special reference to species differences.

Double staining immunohistochemistry was used to investigate the origin and projection of nerves with substance P (SP) immunoreactivity (-IR) in the walls of the major cerebral arteries in two microchiropteran species. In the greater horseshoe bat, most of the cerebral perivascular nerves with SP-IR did not exhibit calcitonin gene-related peptide (CGRP)-IR, but emitted bright immunofluorescence for vasoactive intestinal polypeptide (VIP). In this species, a large number of cell bodies with both SP- and VIP-IR were observed in many cranial ganglia along various branches of the facial and glossopharyngeal nerves. There were no cell bodies immunoreactive for either SP and VIP in the two sensory (trigeminal and upper cervical dorsal root), two sympathetic (superior cervical and stellate), or two vagal (superior and jugular) ganglia. In addition, several thick fiber bundles with both SP- and VIP-IR were present in the wall of the cerebral carotid artery, and descended progressively reaching as far as the middle part of the basilar artery (BA). These and other findings suggest that SP-immunoreactive nerves with VIP-IR but not CGRP-IR, which contribute to the rich innervation of the vertebrobasilar system in the greater horseshoe bat, originate from neurons with the same combination of peptide-IR in the major or local facial or glossopharyngeal parasympathetic ganglia, and enter the cranial cavity along the internal carotid artery. In the bent-winged bat, however, cerebral perivascular SP-immunoreactive nerves, as well as SP-immunoreactive neurons within the trigeminal and upper cervical dorsal root ganglia (uCDRG), showed neither CGRP-IR nor VIP-IR, and were mostly confined to the caudal BA and the vertebral artery (VA). These observations, in addition to the projection of this nerve type to the BA via the VA as fiber bundles, or through the meninges, indicate that the principal source of the cerebrovascular SP-immunoreactive innervation in this species is the uCDRG.

Chemotherapeutic approach to uterine papillary serous carcinoma.

Uterine papillary serous carcinoma (UPSC) is a recently recognized histologic variant of endometrial carcinoma with an aggressive clinical course. This study reviews 7 patients with the diagnosis of UPSC treated at the National Kokura Hospital between 1987 and 1989. The mean age was 60 years. Clinically, 4 patients presented as Stage I, one as Stage II, one as Stage III, and one as Stage IV. Surgery upstaged 28.7% (2/7) of these patients. All 7 patients underwent a staging laparotomy followed by chemotherapy. Deep myometrial invasion was found in 5/7 (72%) of the specimens. Four of these 5 specimens demonstrated up to the uterine serosa. Lymph nodal involvement was observed in 1/4 (25%) of the specimens. Furthermore, positive peritoneal washings were obtained in 4/7 (57%) of the specimens. All patients received an adjuvant combination chemotherapy with cisplatin 50 mg/m2 on day 1, adriamycin 50 mg/m2 on day 1, and ifosfamide 1.0 g/m2 on day 1 to day 5 (PAI). Recurrence and relapse of the disease developed in the 2 patients within 6 months. Two patients had a negative second look operation with no evidence of disease (NED) at 18 months after the initial treatment. Serial determination of the serum CA125 was seen to have great value in monitoring treatment in 4 patients. However, additional accrual and follow-up are needed to determine whether or not PAI therapy has an impact on this disease.

[Platinum-based combination chemotherapy for malignant mixed mesodermal tumor of the ovary: a case report].

Malignant mixed mesodermal tumor (MMMT) of the ovary is a rare neoplasm with a dismal prognosis and therapeutic modalities remain an enigma. The present paper describes our experience with an impressive response utilizing a cis-platinum based combination chemotherapy in a 56-year-old female with this disease and a review of the literature. The patient was treated with PAIE therapy following a cytoreductive surgery. Two further courses of a single-agent chemotherapy of cis-platinum were given. A PR was obtained after completion of the third course and tumor markers showed a prompt fall with a tumor response. Nevertheless, no further therapy was performed because of her refusal. She expired 5 months after diagnosis because of tumor progression.