Four experiments were conducted to determine the composition of a solution suitable for vitrification of immature bovine oocytes. The osmotic and cytotoxic effect of different concentrations (0, 0.25, 0.5 and 1 mol l−1) of nonpermeating solutes (sucrose versus trehalose) were examined. In addition, the effect of permeating cryoprotectants such as glycerol, dimethyl sulfoxide, and propylene glycol (40% w/v) on the viability of oocytes was studied to determine the optimal time of exposure and the most suitable cryoprotectant. Exposure of bovine oocytes to trehalose was less harmful than exposure to sucrose (P < 0.01), and high normospermic fertilization (70%) was achieved after exposure to 0.25 mol trehalose l−. Propylene glycol was chosen as the cryoprotectant for the vitrification of immature bovine oocytes because of its fast permeating rate and its low cytotoxic effect. The composition of this solution (40% propylene glycol and 0.25 mol trehalose l−1 in PBS containing 4% w/v BSA) appeared to be suitable for vitrification, as the fertilization rate of the vitrified oocytes was 37% (36 of 97).
A. Arav, D. Shehu, and M. Mattioli
M. Mattioli, F. Conte, Giovanna Galeati, and E. Seren
Summary. Six lactating sows were injected through an indwelling vena cava cannula with naloxone (2·5 mg/kg body weight) on Day 15 post partum. Blood samples were collected through the cannulas at 10-min intervals for 8 h before and 10 h after naloxone administration. Plasma prolactin and LH concentrations were measured by radioimmunoassay. Naloxone caused a marked suppression of plasma prolactin concentrations lasting 4–6 h. LH concentrations were also affected by naloxone: LH rose to reach maximum values 20–50 min after naloxone treatment. Pretreatment values were recorded 200–300 min after the treatment. These results indicate that endogenous opioids are involved in causing the endocrine patterns occurring during lactation, i.e. high prolactin and low LH concentrations.
Rosella Gaiani, F. Chiesa, M. Mattioli, G. Nannetti, and Giovanna Galeati
Summary. Androstenedione concentrations in both plasma and milk were higher than those of testosterone throughout pregnancy. Testosterone concentration increased during pregnancy; the levels in milk were always lower than in plasma. Androstenedione concentrations increased in plasma and milk during pregnancy but the values were twice as high in milk as in plasma after Day 90. This change in androstenedione distribution suggests that the mammary gland could be active in the uptake of androstenedione from plasma to milk or that the mammary gland itself might synthesize this hormone.
M. Mattioli, G. Galeati, B. Barboni, and E. Seren
Intracellular concentrations of cyclic AMP (cAMP) were measured in pig oocytes maturing in vivo or in vitro. Maturation in vivo was induced with 500 iu hCG administered to gilts treated with pregnant mares' serum gonadotrophin (PMSG). Although PMSG did not affect cAMP concentrations (basal values, 1.69 ± 0.28 fmol per oocyte), hCG induced a transient rise (8.86 ± 1.15 fmol per oocyte 12 h after hCG injection). Similarly, the cAMP concentration rose in oocytes maturing in vitro if the oocytes (surrounded or not by cumulus cells) were co-cultured with the follicle wall in the presence of LH. The same increase in cAMP was obtained when denuded oocytes were co-cultured with mural granulosa cells. Theca cells exhibited only a moderate activity, while cumulus cells were totally ineffective. Granulosa cells exposed to LH lost their stimulating influence after 24 h of culture. In the presence of FSH, cAMP production by the oocyte was unaffected by any type of follicle cell. The role of cAMP in the control of oocyte maturation was investigated using dibutyryl cAMP. The presence of dibutyryl cAMP prevented the resumption of meiosis in a dose-dependent manner, but when it was present during the first 12 h of culture only, meiotic progression was accelerated (0 versus 47% of oocytes had germinal vesicles in groups treated with dibutyryl cAMP and control groups, respectively, after 24 h of culture). The results demonstrate that: (i) cAMP concentrations increase transiently in oocytes before the resumption of meiosis; (ii) increased concentrations of cAMP depend on the stimulation of oocyte adenylyl cyclase, possibly by a soluble factor produced by follicle cells exposed to LH; (iii) the increase in cAMP is probably confined to the first 10–20 h of maturation owing to the progressive reduction of the stimulating influence of LH-treated somatic cells; and (iv) a high concentration of cAMP throughout maturation maintains meiotic arrest and a transient increase may facilitate meiosis.
G. Bono, D. Minori, R. Gaiani, and M. Mattioli
Summary. Peripheral plasma levels of oestrone sulphate, androgens and LH were studied in two mating stallions. A peak of oestrone sulphate was observed 7·5–15 min after exposure to an oestrous female. No variations were recorded in LH concentrations. The concentrations of testosterone, dihydrotestosterone and androstenedione increased similarly after mating and on a day of sexual inactivity.
M Mattioli, A Gloria, A Mauro, L Gioia, and B Barboni
The research has been designed to investigate whether acrosome-reacted spermatozoa can fuse with somatic cells and to check whether this event may involve the molecular machinery implicated in the sperm–egg fusion. Boar spermatozoa were capacitated in vitro and then treated with A23187 to induce acrosome reaction and activate their fusogenic potential. Reacted spermatozoa, loaded with the membrane-permeant fluorescent dye calcein AM, were incubated with plated granulosa cells or cells derived from stable cell lines: CRFK, VERO, and ESK4. The fusion between spermatozoa and somatic cells was revealed by the diffusion of the fluorescent dye from the sperm to the cell as membrane fusion and cytoplasmic continuity between the two cells were established. The involvement of integrin α6 and tetraspanin CD9 in the process of fusion was assessed by carrying out the experiment in the presence of antibodies against these molecules. Moreover, the incidence of fusion displayed by the different cell types used was analyzed in relation to their content in the above molecules assessed by western blot and immunostaining. The role of CD9 was additionally investigated by using CD9-negative cells. The data presented demonstrate that boar spermatozoa can fuse with different somatic cell types derived from different species and the process requires the combined presence of both integrin and tetraspanin molecules on the cell plasma membrane.