G. Siracusa

Meiotic resumption and intracellular cAMP levels in mouse oocytes treated with compounds which act on cAMP metabolism

We have studied the effect of agents known to stimulate adenylate cyclase on spontaneous meiotic resumption in vitro by mouse oocytes. We have found that cholera toxin (CT) (up to 1 microM) and prostaglandin E1 (PGE1) (up to 160 microM) are not able to prevent meiotic resumption, but a clear dose-dependent delay in meiosis resumption was observed during the first 3 h of incubation in medium containing CT or PGE1. The effect became clearer when a small concentration of isobutylmethylxanthine (MIX) (1 microM) was added to the medium. We have also measured the cAMP content of the oocyte: the basal content is 2.1 fmol; this value drops to 0.9 fmol during a 2-h culture period. The decrease is partially prevented if CT is present in the incubation medium, while total cAMP content increases to 3.1 fmol in the presence of 1 mM MIX. The results suggest that isolated mouse oocytes contain toxin and prostaglandin sensitive adenylate cyclase and also an active phosphodiesterase system.

The involvement of calcium in the activation of mammalian oocytes.

Mouse oocytes with cumulus cells intact were parthenogenetically activated following release from the oviduct into calcium-free medium. The proportion of activated oocytes increased with post ovulatory age both for oocytes initially exposed to calcium-free and calcium-containing medium (control). Apart from oocytes released shortly after ovulation (approximately 1 h) when less than 1% of the oocytes from treated and control were activated, activation was always higher in oocytes incubated in calcium-free medium (p less than 0.001). The omission of magnesium from the medium had no effect on the activation response of oocytes obtained approximately 3 h after ovulation but its absence did increase the activation rate of oocytes of later post ovulatory age (approximately 9 h after ovulation) although it was still lower than that obtained with media devoid of calcium. When the extracellular calcium was replaced by other divalent cations (strontium, barium and manganese) high rates of activation were obtained even at post ovulatory times which produced relatively low rates of activation in calcium-free medium alone. Similar results were obtained when hamster oocytes were exposed to all the aforementioned treatments. It is concluded that calcium plays an essential role in the activation of the mammalian oocyte but the mechanism of its action remains obscure. Further development of oocytes activated by calcium-free treatment was limited and was similar to that of oocytes activated in other ways.

Survival of isolated, fully grown mouse ovarian oocytes is strictly dependent on external Ca2+☆

Abstract The presence of Ca 2+ is essential for survival in culture of fully grown oocytes isolated from mouse ovaries but not for survival of small, meiotically incompetent oocytes, metaphase II oocytes, and early embryos. Ninety percent of fully grown ovarian oocytes die within 2 hr when cultured in calcium-free medium (CFM). CFM death does not occur if other cations (1 m M La 3+ or 10 m M Sr 2+ , but not 12 m M Mg 2+ nor 1 m M D-600) replace Ca 2+ in the medium. Sensitivity to CFM is progressively acquired by the oocyte during the growth phase, in parallel with the acquisition of meiotic competence, and is lost after 2 hr of culture in the presence of at least 0.5 m M Ca 2+ . The loss of sensitivity to CFM during in vitro culture is not related to the concomitant spontaneous resumption of meiosis, since the oocyte becomes resistant to CFM even if germinal vesicle breakdown is prevented by the addition of dibutyryl cAMP to the culture medium. Some hypotheses are put forward to explain the peculiar and transient high calcium requirements of fully grown oocytes.

Expression of differentiative traits in the absence of cell fusion during myogenesis in culture

Fusion of myoblasts is inhibited in cultures at low Ca++ concentration (0.44 mM); yet creatine phosphokinase and myokinase activities as well as myosin synthesis and the appearance of post-mitotic myoblasts do not significantly differ from those of control cultures (grown at 1.04 mM Ca++) which undergo cell fusion. When Ca++ concentration is increased to the control value after the second day of culture, fusion occurs very rapidly and it is not inhibited by actinomycin D or cycloheximide. Treatment with 0.06 mM bromodeoxyuridine strongly inhibits creatine phosphokinase activity and myotubes formation. The study of the kinetics of reversal of cell fusion and of creatine phosphokinase activity after removal of the analog, shows that this process is slower than the decrease of the relative content of bromodeoxyuridine incorporated into DNA. The result obtained support the following conclusions: a) the expression of the differentiative characters examined does not require cell fusion; b) the process of myotube formation seems to imply two subsequent stages consisting first of a slow maturative process, which is followed by the actual fusion of cell membranes; the former is Ca++ independent, the latter is Ca++ dependent and does not require RNA or protein synthesis.

Expression and role of PDGF-BB and PDGFR-beta during testis morphogenesis in the mouse embryo

The role played by PDGF in testis morphogenesis is still incompletely understood. The present study investigates the expression and potential role of platelet-derived growth factor-BB (PDGF-BB) and its receptor, PDGF receptor β (PDGFR-β), during mouse testis cord formation, and the possibility that the growth factor may be involved in the migration to the gonad of mesenchymal cells of mesonephric origin. Studies from this laboratory have previously shown that mesenchymal cells that migrate from the mesonephros into the gonad, to form peritubular myoid cells and most of the intertubular cells, can be identified by the presence on their surface of the p75 neurotrophin receptor (p75NTR), and can be isolated to near-purity by immunomagnetic selection with anti-p75NTR antibody. We show here that mesonephric p75NTR(+) cells also bear the PDGFR-β, and are able to migrate and proliferate in vitro in response to PDGF-BB. PDGF-BB is expressed at higher levels in male than female developing gonads, suggesting a role for this factor in testis development. Such a role is further supported by the observation that addition of PDGF-BB to serum-free medium is sufficient to allow organ-cultured male 11.5 days post-coitum urogenital ridges to form testis cords. Finally, we show that mesonephric cell motility and growth induced by exposure to PDGF-BB involve mitogen-activated protein kinases (MAPK) and phosphatidylinositol-3 kinase (PI3-K) pathways, as MAPK inhibitor U0126 and PI3K inhibitor Ly294002 inhibit migration and proliferation in vitro assays. The present findings support the hypothesis that the PDGF/PDGFR system plays a key role in testis morphogenesis in the mouse embryo.

Zona pellucida solubility and cortical granule complements in human oocytes following assisted reproductive techniques

In this study the solubility to alpha-chymotrypsin of the zona pellucida (ZP) of human oocytes and polyploid embryos obtained during various clinical procedures of assisted fertilisation (IVF, ICSI, cyropreservation) was evaluated. The aim of the study was to determine whether changes in ZP solubility occur during such procedures and whether abnormal solubility could be likened to fertilisation failure. Correlation between ZP solubility and cortical granule (CG) density was also studied. The results showed that ZP solubility varied considerably among germinal vesicle or metaphase oocytes obtained from different subjects, but was essentially identical for the oocyte cohort obtained from individual women. On the basis of ZP solubility metaphase oocytes were subdivided into two classes: class I, average ZP dissolution time +/- SE = 24.1+/-0.9 min, n = 28; and class II, 46.7+/-2.0 min, n = 13. Prolonged ZP dissolution times of metaphase oocytes were significantly correlated with a low in vitro fertilisation rate in sibling oocytes. The zonae of fertilised eggs (polyploid embryos) showed long solubilisation times (IVF: 45.3+/-3.4 min, n = 18; ICSI: 48.9+/-2.7 min, n = 19). ZP solubility of oocytes that failed to fertilise was intermediate between that of class I metaphase oocytes and embryos (unfertilised IVF: 33.0+/-2.7 min, n = 13; unfertilised ICSI: 43.0+/-2.4 min, n = 9). A moderate spontaneous ZP hardening occurred when metaphase oocytes were cultured for 24 h. Finally, cryopreservation of unfertilised oocytes caused hardening of their ZP, with dissolution times that were comparable to those found in fertilised eggs (49.5+/-2.3 min, n = 10). In most cases, an inverse correlation was found between ZP dissolution time and CG density (longer solubilisation times corresponding to lower CG density). ZP hardening caused by cryopreservation, however, was not associated with a significant reduction in CG density in most of the oocytes examined.

Differential effects of metabolic inhibitors on early development in the mouse embryo, at various stages of the cell cycle☆

Abstract Mouse embryos were explanted after different intervals during the interphase between the first and second cleavage and allowed to develop in culture in the presence of actinomycin D or puromycin. The treatment with the inhibitors during the early part of the cell cycle causes complete arrest of cleavage, while treatment during the late part of the cell cycle is completely ineffective. These results suggest that the cleavage is regulated by RNA and protein molecules which need to be synthesized during each cell cycle.

Pyruvate and lactate production by cultured Sertoli cells, fibroblasts and muscle satellite cells, and the effect of hormonal and dcAMP stimulation

Isolated male germ cells survive in culture if medium is supplemented with adequate energy substrates such as lactate or pyruvate. The purpose of the present study was to investigate if cultured Sertoli cells release in the medium lactate or pyruvate and if this production is affected by FSH or dcAMP treatment. We have also studied if the ability to produce lactate and pyruvate is shared by other cell types. The results show that 1) the two metabolites are released from germ-cell-free rat Sertoli cell monolayers, and their release is stimulated by hormone or dcAMP 2) other cell types of mesodermic origin release more lactate and pyruvate than Sertoli cells, but are not stimulated by FSH or dcAMP.

Local anesthetics and phenothiazine tranquilizers induce parthenogenetic activation of the mouse oocyte

Abstract Incubation of recently ovulated mouse oocytes in various concentrations of local anesthetics and phenothiazine tranquilizers initiates the completion of meiosis and the formation of pronuclei. The potency with which these drugs induce oocyte activation is comparable to their relative ability to displace Ca 2+ from artificial lipid membranes. Oocyte activation induced by these drugs is inhibited by Ca 2+ . Perturbation of the plasma membrane of the oocyte by these drugs may displace Ca 2+ , which is responsible for the integrity of the microtubule-microfilament system that normally maintains the ovulated mammalian oocyte blocked at the Metaphase II of meiosis until sperm penetration.

Fertilization-induced changes in concanavalin A binding to mouse eggs☆

Abstract The binding of concanavalin A (ConA) to zona-free unfertilized and fertilized mouse eggs has been investigated using tritiated ConA. At low lectin concentrations (1–5 μg ml−1) the fertilized egg shows a higher affinity for [3H]ConA than does the unfertilized egg. In saturation conditions, however, unfertilized and fertilized eggs show the same binding capacity (1.55 × 108 ConA molecules/egg). The results indicate that ConA-binding sites change qualitatively following fertilization; possible connections between this change and other fertilization-induced changes in the egg surface are discussed.