Effects of beta-endorphin and Naloxone on in vitro maturation of bovine oocytes.

Bovine cumulus-oocyte complexes (COCs) and mural granulosa cells express the mRNA coding for the micro-opioid receptor. The addition of beta-endorphin (beta-end) to oocytes cultured in hormonally-supplemented in vitro maturation (IVM) medium had no effect on the rates of oocytes reaching the metaphase II (MII) stage, but significantly decreased the maturation rate (P < 0.05) and arrested oocytes at metaphase I (MI) after culture in hormone-free medium (P < 0.001). Naloxone (Nx) reverted this inhibitory effect of beta-end. Moreover, Nx "per se" showed a dose-dependent dual effect. When added at high concentration (10 x (-3) M), it significantly reduced the rate of oocytes in MII (P < 0.001), thus increasing the rate of oocytes arrested in MI. However, Nx added at low concentration (10 x (-8) M) significantly increased oocyte maturation (P < 0.001). High concentration of Nx induced an increase in both intracellular calcium concentration ([Ca(2+)](i)) and in the activity of the mitogen-activated protein kinase (MAPK) also called extracellular-regulated kinase (ERK) in cumulus cells of bovine COCs. Blocking the rise in [Ca(2+)](i) with the calcium chelator acetoxymethylester-derived form of bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) reversed the Nx-dependent inhibition of meiotic maturation observed at high Nx concentrations. Whereas blocking ERK with the MAPK/ERK kinase (MEK) inhibitor, PD98059, had no effect on this process. Therefore, we concluded that the mocro-opioid receptor, by inducing [Ca(2+)](i) increase, participates in the cumulus-oocyte coupled signaling associated with oocyte maturation.

Influence of oocyte collection technique on initial chromatin configuration, meiotic competence, and male pronucleus formation after intracytoplasmic sperm injection (ICSI) of equine oocytes.

There is a great variability in the success of horse oocyte maturation and fertilization among laboratories. This study was conducted to determine if the meiotic and developmental competence of horse oocytes could be dependent on the method of oocyte collection, i.e., aspiration of follicular fluid with a vacuum apparatus, or opening follicles and scraping the granulosa layer. Horse oocytes were recovered from abattoir ovaries by aspiration or scraping and classified as having compact (Cp), expanded (Ex), or partial (P) cumuli. In Experiment 1 (Part A in May and Part B in October), oocytes were fixed immediately after collection to assess whether the collection method influenced the initial chromatin configuration of oocytes. In Experiment 2, in vitro maturation rates of oocytes recovered by aspiration or scraping were compared. In Experiment 3, oocytes were matured in vitro and submitted to intracytoplasmic sperm injection (ICSI). Initial chromatin configuration differed according to collection method in that there was a significantly higher prevalence of diffuse chromatin within the germinal vesicle in oocytes recovered by scraping than in oocytes recovered by aspiration (29/87, 33% and 28/166, 17%, respectively; P < 0.01). Maturation of oocytes to metaphase II did not significantly differ between scraped and aspirated oocytes (56/101, 55.4 % vs. 65/106, 61.4%, respectively). The overall pronucleus formation rate after ICSI of oocytes recovered by scraping was not significantly different than that of oocytes recovered by aspiration (50/99, 52.6% vs. 50/85, 68.5 %, respectively); however, the rate of abnormal fertilization was significantly higher for oocytes collected by aspiration (14/73, 19% vs. 6/94, 6%, respectively; P <0.05). These results demonstrate that the collection method affects the population of recovered oocytes and may contribute to differences in results observed among laboratories working with horse oocytes.

Toxic effects of the mycotoxin zearalenone and its derivatives on in vitro maturation of bovine oocytes and 17 beta-estradiol levels in mural granulosa cell cultures.

Moulds parasites of livestock foodstuffs alter the quality of grains by synthesizing mycotoxins. Zearalenone (ZEA) and its derivatives (alpha- and beta-zearalenol, zeranol, taleranol and zearalanone) are produced by fungi of the genus Fusarium and, after ingestion via contaminated cereals, may lead to fertility disturbances and other reproductive pathologies. Zearalenone, alpha-zearalenol and zearalanone were tested, at levels ranging from 0.3 to 30 microg/ml, in order to evaluate the effect on the in vitro maturation (IVM) rate of bovine oocytes and on the formation of 17 beta-estradiol in supernatants of mural granulosa cells (GC) cultures. These compounds induced dose-dependent oocyte maturation delay and chromatin abnormalities. Maturation of oocytes to metaphase II (M II) was inhibited in oocytes cultured in the presence of 30 microg/ml ZEA, alpha-zearalenol or zearalanone, with a significant increase in chromatin abnormalities occurring in the presence of ZEA (P<0.05) and alpha-zearalenol (P< 0.001). In preliminary trials on 17 beta-estradiol formation, at the same testing concentration, higher levels of 17 beta-estradiol were found in the presence of alpha-zearalenol (mean value 1.6 ng/ml) with respect to ZEA and zearalanone (mean estradiol concentrations of 0.06 and 0.5 ng/ml, respectively). These data demonstrate a negative effect of ZEA and its derivatives on meiotic progression of bovine oocytes, possibly attributable to a toxic mechanism not related to the binding affinity of these compounds to estrogen receptor sites, and support previous observations that alpha-zearalenol acts as a stronger estrogenic inducer than the original molecule (ZEA).

Effects of fetuin on zona pellucida hardening and fertilizability of equine oocytes matured in vitro.

In vitro fertilization (IVF) has had poor success in the horse, a situation related to low rates of sperm penetration through the zona pellucida (ZP). Zona pellucida hardening (ZPH) is seen in mouse and rat oocytes cultured in serum-free medium. The hardened ZP is refractory to sperm penetration. Fetuin, a component of fetal calf serum, inhibits ZPH and allows normal fertilization rates in oocytes cultured in the absence of serum. We evaluated whether fetuin is present in horse serum and follicular fluid (FF) and whether fetuin could inhibit ZPH in equine oocytes matured in vitro, thus increasing sperm penetration during IVF. The presence of fetuin in equine serum and FF was confirmed by immunoblotting. Oocytes submitted to in vitro maturation (IVM) in medium containing fetuin were used for ZPH assay or IVF. Intracytoplasmic sperm injection (ICSI) was carried out as a control procedure. The presence of fetuin during IVM did not affect the rate of maturation to metaphase II. Maturation of oocytes in the presence of fetuin reduced ZPH in a dose-dependent manner. After both IVF and ICSI, there was no significant difference in oocyte fertilization between fetuin-treated and untreated oocytes. The fertilization rate was significantly higher after ICSI than after IVF, both in fetuin-treated and in untreated oocytes. In conclusion, fetuin reduced ZPH in equine oocytes but did not improve sperm penetration during IVF. This implies that, in the horse, "spontaneous" ZPH is unlikely to be the major factor responsible for inhibiting sperm penetration in vitro.

Vasculitic neuropathy associated with beta-haemolytic streptococcal infection: a case report.

Vasculitic neuropathy is rarely associated with a definable collagen vascular disease. Peripheral neuropathy may be the sole manifestation of vasculitis, and the aetiology is frequently unknown. We here report the case of a woman presenting mononeuritis multiplex, whose sural nerve biopsy was diagnostic of necrotizing vasculitis. There was serological evidence of preceding beta-haemolytic streptococcal infection. We assume that vasculitic neuropathy can be included among the possible sequelae of streptococcal infections.

[Combined outpatient surgical-cryotherapeutic treatment of anal fissures. Our experience]. / Il trattamento combinato chirurgico-crioterapico ambulatoriale della ragade anale. Nostra esperienza.

The authors describe their technique and their experience of ambulatory surgical-cryotherapeutic combined, treatment of anal fissure. The data were observed in 35 patients (medium age 37.5); in 16 cases, previous treatments gave no benefit. The surgical treatment was the lateral internal close sphincterotomy according to Notaras, with local anesthesia (personal technique), followed by a fissure curettage with a N protosside cryosound. The results confirm the well known effectiveness of lateral internal sphincterotomy and the validity of ambulatory treatment and of cryotherapy.

[The evolutionary principles of inguinal hernioplasty through our personal experience]. / Principi evolutivi dell'ernioplastica inguinale attraverso la nostra esperienza personale.

Inguinal hernia is a multi-factorial pathologic condition. A constitutional weakness of transversalis fascia, probably genetically transmitted, represents a fundamental cause. Hernioplasty has evolved year by year from the "Bassini" to the "tension-free" techniques with Marlex or Prolene prosthesis, on the rational base of no tension on constitutionally weak structures. The data reported confirmed the absolute validity of "tension-free" hernioplasty.

Intracytoplasmic sperm injection (ICSI) versus conventional IVF on abattoir-derived and in vitro-matured equine oocytes.

Conventional IVF as well as several assisted microfertilization techniques have shown limited success in the horse. After recent positive results achieved with intracytoplasmic injection of a single spermatozoon (ICSI) in human IVF, we chose to try the method in the horse. We compared conventional IVF to ICSI by fertilization rates of oocytes with compact and expanded cumuli and by developmental potential of the resulting embryos. Cumulus-oocyte complexes (COCs) were obtained by aspirating the follicular fluid from the ovaries of slaughtered mares. Complexes showing complete cumulus investment, either compact or expanded, were randomly assigned to IVF or ICSI trials and separately cultured for IVM. Frozen-thawed stallion spermatozoa were prepared for IVF with a swim-up procedure conducted in Talp-Hepes with heparin or for ICSI in Earle's balanced salt solution (EBSS) supplemented with human serum albumin (HSA). Oocytes for IVF were partially decumulated by pipetting, whereas those for ICSI were totally denuded with 80 UI/ml hyaluronidase. Oocytes were fixed, stained and examined for signs of fertilization the day after IVF or ICSI. The percentage of normally fertilized oocytes showing 2 pronuclei or cleavage was significantly higher with ICSI than IVF (29.8%, 17/57 vs 8.7%, 9/103 ; P < 0.01). Significantly higher fertilization rates were observed in oocytes retrieved with an expanded cumulus when submitted to ICSI procedure as compared with IVF (52.2%, 12/23 vs 17.1%, 6 35 ; P < 0.01), whereas in oocytes recovered with a compact cumulus, fertilization rates were low (14.7%, 5/34 with ICSI and 4.4%, 3 68 with IVF; NS). Embryonal development did not occur after culture following IVF, as indicated by absence of cleavage in any of the 93 inseminated oocytes. Following ICSI, 7 of 55 injected oocytes cleaved, 5 of which had shown expanded cumuli; of the 5, 2 were at the 16-cell stage and one each at the 8-, 3- and 2-cell stage, respectively. The other 2 fertilized oocytes, originating from compact cumuli, reached 4- and 8- cell stages, respectively. These results indicate that ICSI can be applied successfully to in-vitro matured equine oocytes to increase the fertilization rates. In addition, it seems that in vitro cytoplasmic maturation of oocytes issuing from a compact cumulus may not be complete enough to lead to a successful fertilization and that ICSI may be a tool to evaluate ooplasmic maturation.

[Clinico-diagnostic aspects and therapeutic considerations of the combined surgical and radiotherapeutic approach in 2 cases of sacrococcygeal chordoma: our experience]. / Aspetti clinico-diagnostici e considerazioni terapeutiche dell'approccio combinato chirurgico e radioterapico in due casi di cordoma sacro-coccigeo: nostra esperienza.

Chordomas are rare tumours arising from embryonic notochord tissue remnants. The commonest affected segment is the sacrum. This localization may present diagnostic and therapeutic problems. Two cases of chordoma treated by surgery and radiation are reported.

[Brucellosis with osteo-muscular localization. A case report of a not rare pathological condition]. / Brucellosi a localizzazione osteo-muscolare. Un caso come condizione patologica non infrequente.

Brucellosis may show itself only through its bone and muscle complications, especially in the lumbar region, such as vertebral colliquation or psoas abscess. The authors describe a case of brucellosis (56 year old male, butcher) with lumbar bone and muscle involvement. The first symptom was a persistent pain of the right lumbar region, with irradiation to the homolateral leg. At admission the patient showed a lumbar cutaneous fistula with pus-like secretion. Quick diagnosis and therapy can prevent irreparable damage, even when the symptoms are silent.

Effects of follicular fluid supplementation of in-vitro maturation medium on the fertilization and development of equine oocytes after in-vitro fertilization or intracytoplasmic sperm injection.

The aim of this study was to compare the effect of the addition of follicular fluid (FF) collected from preovulatory follicles with that of oestrous mare serum (EMS) (acting as the control) to TCM-199 medium on the in-vitro maturation, fertilization and development of equine cumulus-enclosed oocytes. Oocytes (<30 mm in diameter) were obtained from the ovaries of slaughtered mares. After in-vitro maturation in the presence of the two supplements, their fertilization, cleavage and developmental potential were compared after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) using frozen-thawed spermatozoa. Follicular fluid did not increase the maturation of oocytes to metaphase II stage compared to control. After IVF, there was no difference in fertilization rates between FF-supplemented oocytes and controls (7/87, 8.4% of oocytes showing two pronuclei with FF versus 7/116, 6% with EMS; not significant). However, after ICSI, FF-supplemented oocytes showed significantly increased normal fertilization (32/85, 37.6% of two-pronuclear oocytes) and developmental potential (15/31, 48% cleavage) compared to the control oocytes (7/47, 14.9%, P < 0.01; and 2/48, 4%, P < 0.01, respectively). Overall, ICSI resulted in increased fertilization rates compared to IVF, regardless of the presence or absence of FF (39/132, 29.5% with ICSI versus 14/203, 6.9%). These results suggest that follicular fluid supplementation may improve the maturity of equine cumulus-enclosed oocytes sufficiently for the successful use of ICSI, but not sufficiently for normal sperm-egg interaction occurring during IVF.

[Adenocarcinoma of the anal glands. Description of a clinical case and review of the literature]. / Adenocarcinoma delle ghiandole anali. Descrizione di un caso clinico e revisione della letteratura.

Adenocarcinoma of the anal glands is a rare slow-growing tumor with a more favorable prognosis compared with colorectal adenocarcinoma, especially if an early diagnosis is established. Clinical symptoms of this disease, often associated with a fistula in ano as in the reported case, include: perianal pain, rectal bleeding and presence of perianal mass. Also perianal Paget's disease may be a not rare association with adenocarcinoma of the anal glands. We believe, therefore, that a histological examination of the resected fistulas in ano should be performed, in searching for the presence of mucinous granules. In addition, it's very important to carry out a careful examination of those patients presenting pruritus ani or eczematous lesions of the anal region. In fact adenocarcinoma of the anal glands may be due to a chronic irritation of the epithelium over a period of years. Radiation therapy and chemotherapy have proved not to provide survival benefit in the treatment of this disease; the same result is obtained with a local excision of the lesion. The only chance for cure, therefore, is early diagnosis followed by radical operation. Miles abdominoperineal resection represents the approach of choice we have adopted for our patient. A radical groin dissection should be carried out only if there are metastases to the inguinal nodes. If necessary, abdomino-perineal resection may be folowed by adjuvant irradiation.

[Our experience with toracic outlet syndrome]. / La nostra esperienza nelle sindromi dello stretto toracico superiore.

The Authors report their experience in 290 cases of thoracic outlet syndrome (TOS) and 71 cases of Paget-Schroetter syndrome, that is a condition due to thrombosis of the subclavian and/or axillary vein. They point out that diagnosis is mainly clinical-instrumental and the ulnar nerve conduction velocity (UNCV) test is of fundamental importance. The Authors emphasize how the good results obtained could be related to the complete removal of the first rib and to the axillary approach that they strongly support.

[Sclerosing adenocarcinomas of the hepatic hilum]. / Gli adenocarcinomi sclerosanti dell'ilo epatico.

The authors report their series of 7 adenocarcinomas of the hepatic hilum. Five patients with a stage IV tumor underwent palliative surgery while the remaining 2 patients underwent radical surgery. One patient died and 3 reported postoperative complications. Overall survival was 2 years and 6 months: these results can be considered satisfactory taking into account the advanced stage and the bad prognosis of this type of tumor. The aim of a better quality of life may represent a reasonable indication to surgery.

[Esophagectomy without thoracotomy: indications and our experience]. / Esofagectomia senza toracotomia: indicazioni e nostra esperienza.

In this paper the Authors report their experience of Orringer operation in 12 patients with oesophageal cancer observed from 1978 to 1992, and stress the possibility to extend the indications to Akijama oesophagectomy without thoracotomy for the treatment of malignant tumors of the entire oesophagus.

In vitro maturation and fertilization of equine oocytes recovered during the breeding season.

The aim of this study was to develope an efficient and reproducible procedure for in vitro maturation (IVM) and fertilization (IVF) in the horse. Cumulus-oocyte complexes (COCs) recovered from the ovaries of mares slaughtered during the breeding season were morphologically evaluated, and those showing a compact cumulus and homogeneously appearing cytoplasm were selected for culture. Effects on the maturation of estrous mare serum (EMS) versus estrous cow serum (ECS) as medium supplement were also evaluated (Experiment 1). In Experiment 2, the fertilization of in vitro matured oocytes with frozen-thawed semen separated by swim-up and treated with heparin was carried out to determine the effects on fertilization of 1) increasing sperm concentrations (1x 10(6), 5 x 10(6) and 1 x 10(7)sperm cells/ml), 2) IVM medium supplementation with EMS or ECS and 3) partial cumulus mass removal before insemination. Forty-nine percent of the collected oocytes (335 683 ) showed a compact cumulus and homogeneous ooplasm and thus were selected for culture. In Experiment 1, high nuclear maturation rates were observed in both EMS (82%,32 39 ) and ECS (87.5%,56 64 ) groups, with no statistically significant difference. In Experiment 2, the percentage of normal fertilization (2 polar bodies, 2 pronuclei and sperm tail) was similar for all 3 tested sperm concentrations (12.9%,4 31 ; 15.2%,9 59 and 15.5%,9 58 ). No advantage in using the homologous serum in IVM medium was noted in terms of fertilization (12.2%, 5 41 with EMS vs 12.9%, 4 31 for ECS). However, significantly higher fertilization rates were obtained after partial cumulus removal compared with that of oocytes fertilized with a whole cumulus (32.6%, 14 43 vs 12.2%, 5 41 ; P < 0.05). The incidence of polyspermic fertilization was low under all culture conditions (0 to 2.4%). In a replicate in which the oocytes fertilized after the cumulus removal were further cultured for 72 h two embryos, one at the 2-cell stage and the other at the 4-cell stage, could be obtained. These results indicate that, in the horse, the cumulus can be partially removed to increase the fertilization of compact-cumulus oocytes recovered during the breeding season using frozen-thawed, heparin-treated semen.

[Therapeutic strategies in the treatment of esophageal cancer: our experience]. / Strategie terapeutiche nel trattamento del cancro dell'esofago: nostra esperienza.

Esophageal cancer has a poor prognosis. The Authors in reviewing esophageal carcinogenis stress the importance of an early diagnosis to improve surgical results and compare their experience with those of other Authors.

[Role of surgery in the treatment of gastroduodenal ulcer]. / Ruolo della chirurgia nel trattamento dell'ulcera gastroduodenale.

Medical approach to gastroduodenal ulcer has dramatically changed with the advent of anti-H2 drugs. There is still a role for surgery, but it has been confined to the treatment of the complications (perforation, stenosis and some cases of haemorrhage). In this paper the Authors collected the data of 43 patients (27 males and 16 females, mean age 45), operated between July 1988 and December 1992; 12 patients were operated for pyloric stenosis, 16 for gastrointestinal bleeding and 15 for perforation. Surgery was accomplished as an emergency procedure in 20 cases, delayed emergency procedure in 4 cases and elective procedure in 19 cases. 39 out of 43 patients were under anti-H2 treatment for a previous diagnosis of peptic ulcer. In conclusion, anti-H2 therapy, even correctly carried on, may not be effective in avoiding possible complications. This goal may be obtained in patients under omeprazole treatment.

[Femoro-femoral crossover bypass in monolateral obstructions of the iliac artery]. / Il bypass femoro-femorale cross over nelle ostruzioni monolaterali dell'arteria iliaca.

The Authors report the result of their ten-years experience with femoro-femoral crossover bypass in 26 patients affected with unilateral occlusion of the iliac artery. This bypass is an easy and quick procedure, useful in both short and long term. The patency rate after 58 months is 84.6%. Thus, its use indicated in a high number of patients, not only for a limb salvage treatment of a disabling claudication. The indications for this bypass can be extended to low surgical risk subjects, and it does not have to be considered only for the high-risk patients, instead of major surgery procedure on the aorto-iliac axis. The minimal necessary conditions of the limbs for performing the femoro-femoral crossover bypass are a pressure gradient of 35 mmHg and the angiographical demonstration of the unilateral occlusion. If the contralateral axis appears patent, but there are also multiple atherosclerotic sites, we consider other bypasses as therapeutical choices: aorto-bifemoral, ilio-femoral and the femoro-femoral cross-over itself; in the latter case we previously perform a transluminal angioplasty or an endarterectomy of the donor iliac artery. In these situations is essential to evaluate of the benefit/risk rate for every single patient.