Sandra Cecconi

In vitro growth of preantral follicles isolated from cryopreserved ovine ovarian tissue

Abstract In the present study, we compared the in vitro development of sheep preantral follicles obtained from unfrozen or frozen ovarian cortex. After thawing, follicles stored by a slow-freezing protocol with dimethyl sulfoxide (DMSO) or ethylene glycol (EG) were mechanically isolated and cultured for 10 days. After 1 day, approximately 50% and 34% of the DMSO and EG follicles, respectively, showed overt signs of degeneration, as confirmed by histological analysis. Follicles that survived thawing grew and formed antral-like cavities, without significant differences among experimental groups. However, the percentages of healthy oocyte-cumulus cell complexes (OCCs) retrieved from in vitro-grown follicles, as well as estradiol, were lower in DMSO than in EG or unfrozen follicles. Although cryopreservation did not cause appreciable differences in follicle morphological aspects, frozen OCCs showed lower metabolic cooperativity levels, as determined by [3H]uridine uptake. During culture, oocytes increased in diameter, but the percentage of germinal vesicle stage-arrested oocytes showing a rimmed chromatin configuration was significantly lower in the frozen groups. Our results indicate that cryopreserved sheep preantral follicles underwent growth in vitro but that freezing/thawing specifically affected gap junctional permeability and impaired the progression of regulative processes, such as the acquisition of a specific oocyte chromatin configuration. Moreover, because the cryoprotectant toxicity test excluded the occurrence of direct cellular damage, this method allowed us to discriminate the effects exerted by different cryoprotectants during the cryopreservation procedure on whole-follicular development.

The role of Akt signalling in the mammalian ovary

The serine/threonine protein kinase Akt is involved in many cellular processes including cell growth, survival, proliferation and metabolism. Akt activity is modulated downstream of phosphatidylinositol-3-kinase (PI3K) in response to different extracellular stimuli. In the mammalian ovary, Akt collaborates with other kinases in the regulation of coordinate follicle and oocyte development. Akt determines the pool of primordial follicles and the transition from quiescent to growing phase. In addition, the kinase modulates granulosa cell apoptosis throughout folliculogenesis. In oocytes Akt participates in the control of meiosis resumption and, at metaphase II stage, regulates polar body emission and spindle organization. Its inhibition negatively affects preimplantation embryo development. As a consequence of such a central role, Akt dysregulation is associated with several human diseases including infertility and ovarian cancer.

The fungicide mancozeb induces toxic effects on mammalian granulosa cells.

The ethylene-bis-dithiocarbamate mancozeb is a widely used fungicide with low reported toxicity in mammals. In mice, mancozeb induces embryo apoptosis, affects oocyte meiotic spindle morphology and impairs fertilization rate even when used at very low concentrations. We evaluated the toxic effects of mancozeb on the mouse and human ovarian somatic granulosa cells. We examined parameters such as cell morphology, induction of apoptosis, and p53 expression levels. Mouse granulosa cells exposed to mancozeb underwent a time- and dose-dependent modification of their morphology, and acquired the ability to migrate but not to proliferate. The expression level of p53, in terms of mRNA and protein content, decreased significantly in comparison with unexposed cells, but no change in apoptosis was recorded. Toxic effects could be attributed, at least in part, to the presence of ethylenthiourea (ETU), the main mancozeb catabolite, which was found in culture medium. Human granulosa cells also showed dose-dependent morphological changes and reduced p53 expression levels after exposure to mancozeb. Altogether, these results indicate that mancozeb affects the somatic cells of the mammalian ovarian follicles by inducing a premalignant-like status, and that such damage occurs to the same extent in both mouse and human GC. These results further substantiate the concept that mancozeb should be regarded as a reproductive toxicant.

Expression localisation and functional activity of pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal polypeptide and their receptors in mouse ovary

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) positively affect several parameters correlated with the ovulatory process. PACAP is transiently expressed in rat preovulatory follicles, while VIP is present in nerve fibres at all stages of development. These two peptides act by interacting with three types of receptors: PACAP type I receptor (PAC1-R), which binds with higher affinity to PACAP, and two VIP receptors (VPAC1-R and VPAC2-R), which bind to PACAP and VIP with equal affinity. The aim of the present study was to characterise the PACAP/VIP/receptor system in the mouse ovary. Results obtained by RT-PCR, immunohistochemistry and in situ hybridisation showed that PACAP was transiently expressed in granulosa cells of preovulatory follicles after human chorionic gonadotrophin (hCG) stimulation, while VIP mRNA was never observed. All the receptors were present in 22-day-old untreated mice. In preovulatory follicles, PAC1-R was expressed both in granulosa cells and in residual ovarian tissue but was stimulated by hCG mainly in granulosa cells; VPAC2-R was present in both the cell compartments and was only mildly stimulated; VPAC1-R was present mainly in the residual ovarian tissue and was downregulated by hCG. PACAP and VIP were equipotent in inhibiting apoptosis in granulosa cells, confirming the presence of functional PACAP/VIP receptors. The contemporary induction by hCG of PACAP and PAC1-R in granulosa cells of preovulatory follicles suggests that, also in mouse ovary, PACAP may play a significant role around the time of ovulation. Moreover, the presence of PACAP/VIP receptors in the untreated ovary suggests a possible role for PACAP and VIP during follicle development.

The Effects of the Endocrine Disruptors Dithiocarbamates on the Mammalian Ovary with Particular Regard to Mancozeb

Many human-made chemicals are called endocrine disruptors (EDs) because they have the potential to disrupt endocrine functions in exposed organisms. Many EDs can disrupt hormonal homeostasis by interfering with hormone receptor recognition, binding and activation, while others act by still unknown mechanisms. Among the EDs specifically affecting the female reproductive system, those with steroidogenic/antisteroidogenic effects have been extensively studied and the mechanisms of toxicity clarified also at molecular level. For many others, information is restricted to few epidemiological data and in vivo/in vitro experiments with animal models. This is the case of the dithiocarbamates, and in particular of the fungicide mancozeb, an ethylenedithiocarbamate widely used to protect fruit and vegetables, ginseng included, because of its low acute toxicity in humans. Although the mechanism(s) by which mancozeb may specifically act on female reproductive organs are largely unknown, data on experimental animals in vivo have demonstrated that the fungicide can induce several disturbances on estrus cycle. When used in vitro at concentrations considered too low to cause human health injuries, the fungicide impairs mouse embryo development and meiotic spindle assembly. The possibility that the female germ cell (the oocyte) could be a specific target of mancozeb suggests a role for this fungicide as probable inductor of infertility also in exposed human populations.

Mammalian oocyte growth in vitro is stimulated by soluble factor(s) produced by preantral granulosa cells and by Sertoli cells

We have evaluated the possibility that mouse oocyte growth in vitro could be achieved under the influence of soluble compound(s) released by different somatic cell types. For this purpose, zona‐free denuded oocytes from 12‐day‐old mice were cultured on monolayers of NIH‐3T3 fibroblasts, which are able to establish gap junctional communications with them, in the presence or absence of media conditioned by preantral granulosa cells or by Sertoli cells, plated at increasing concentrations from 0.3–1 × 106 ml−1 cells. After 3 days, no increase in vitellus diameter was recorded from fibroblast‐coupled oocytes maintained in culture medium or in the presence of media conditioned by 0.3 × 106 ml−1 Sertoli cells. By contrast, increasing proportions of coupled oocytes grew, provided the continuous presence of media conditioned by 0.5 or 1 × 106 ml−1 Sertoli cells, or by 0.3, 0.5, and 1 × 106 ml−1 preantral granulosa cells. Since the ligand of c‐kit, the growth factor KL, promotes the growth in vitro of oocytes cultured in follicles from 8‐day‐old mice, an antibody against mouse KL was used to evaluate whether in our culture conditions KL might also be responsible for the growth of oocytes from 12‐day‐old mice. No inhibition of growth was evident in oocytes cultured directly on preantral granulosa or Sertoli‐cell monolayers. Furthermore, the growth of fibroblast‐coupled oocytes cultured in media conditioned by preantral granulosa cells was not significantly affected by the presence of this antibody during culture. By contrast, a high percentage of oocytes cultured on fibroblasts in the presence of media conditioned by Sertoli cells showed a significant inhibition of growth and no metabolic cooperativity. It was concluded that, besides KL, other bioactive factor(s) released by either preantral granulosa or Sertoli cells can induce a significant stimulation of mouse oocyte growth in vitro.

Characterization, Expression, and Functional Activity of Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors in Human Granulosa-Luteal Cells

CONTEXT Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are found in the ovary of mammalian species, although nothing is known about the possible role of PACAP and VIP in the human ovary. OBJECTIVE We investigated the expression of PACAP and PACAP/VIP receptors in human granulosa-luteal (GL) cells obtained from consenting in vitro fertilization patients attending a private fertility clinic and assessed a possible antiapoptotic effect of these molecules. MAIN OUTCOME MEASURES We measured the expression of PACAP and PACAP/VIP receptor mRNAs in GL cells in response to FSH or LH, as well as the effects of PACAP and VIP on apoptosis. We also evaluated the levels of procaspase-3 in GL cells cultured in the absence of serum. RESULTS After 7 d in culture, GL cells displayed increased responsiveness to FSH and LH (100 ng/ml). FSH and LH promoted PACAP expression, LH doing so in a time-dependent fashion. VIP receptor (VPAC1-R and VPAC2-R) mRNAs were also induced by gonadotropin stimulation. Although PACAP receptor (PAC1-R) mRNA was barely detectable, Western blot analysis revealed its presence. The apoptotic effect of serum withdrawal from the culture environment was reverted by both PACAP and VIP. Both peptides showed the ability to reverse a decrease in procaspase-3 levels induced by culture in the absence of serum. CONCLUSIONS PACAP and VIP appear to play a role in maintenance of follicle viability as a consequence of the antiapoptotic effect. Further studies are warranted to evaluate the respective roles of PACAP and VIP in ovarian physiology and to identify their mechanism of action.

Human embryo development and pregnancies in an homologous granulosa cell coculture system.

AbstractPurpose: Our purpose was to determine the effects of thecoculture of embryos on human granulosa cells (GCs) inpatients in the first cycle of IVF-ET treatment and in patientswith repeated implantation failures and to investigate thepresence of specific proteins in a 48-hr GC conditionedmedium and the GC ultrastructural characteristics. Methods: Eighteen patients with tubal or idiopathicinfertility were enrolled in this study: 7 patients (Trial 1) were inthe first cycle of IVF-ET treatment and 11 patients (Trial 2)had repeated implantation failures (one to five). Embryosfrom each patient were cocultured randomly either onhomologous granulosa cells or on a conventional culturemedium. Results: At the end of the coculture period (day 5 or 6),50% of the embryos (Trial 1) reached the blastocyst stage,with respect to 35% in Trial 2. The pregnancy rate perretrieval was 14.2 and 9%, respectively, in Trial 1 and inTrial 2. Many conditioned media showed proteins of 24–29kDa. and some of them showed additional proteins of 90kDa. The ultrastructural analysis of GCs showed healthy,metabolically active, protein-synthesizing, and mostlysteroidogenic cells. Conclusions: GC cultures improve embryo development butnot pregnancy rates both in Trial 1 and in Trial 2.

Ultrastructure of isolated mouse ovarian follicles cultured in vitro.

BackgroundIn vitro maturation of ovarian follicles, in combination with cryopreservation, might be a valuable method for preserving and/or restoring fertility in mammals with impaired reproductive function. Several culture systems capable of sustaining mammalian follicle growth in vitro have been developed and many studies exist on factors influencing the development of in vitro grown oocytes. However, a very few reports concern the ultrastructural morphology of in vitro grown follicles.MethodsThe present study was designed to evaluate, by transmission and scanning electron microscopy, the ultrastructural features of isolated mouse preantral follicles cultured in vitro for 6 days in a standard medium containing fetal calf serum (FCS). The culture was supplemented or not with FSH.ResultsThe follicles cultured in FCS alone, without FSH supplementation (FCS follicles), did not form the antral cavity. They displayed low differentiation (juxta-nuclear aggregates of organelles in the ooplasm, a variable amount of microvilli on the oolemma, numerous granulosa cell-oolemma contacts, signs of degeneration in granulosa cell compartment). Eighty (80)% of FSH-treated follicles formed the antral cavity (FSH antral follicles). These follicles showed various ultrastructural markers of maturity (spreading of organelles in ooplasm, abundant microvilli on the oolemma, scarce granulosa cell-oolemma contacts, granulosa cell proliferation). Areas of detachment of the innermost granulosa cell layer from the oocyte were also found, along with a diffuse granulosa cell loosening compatible with the antral formation. Theca cells showed an immature morphology for the stage reached. Twenty (20)% of FSH-treated follicles did not develop the antral cavity (FSH non-antral follicles) and displayed morphological differentiation features intermediate between those shown by FCS and FSH antral follicles (spreading of organelles in the ooplasm, variable amount of microvilli, scattered granulosa cell-oolemma contacts, signs of degeneration in granulosa cell compartment).ConclusionsIt is concluded that FSH supports the in vitro growth of follicles, but the presence of a diffuse structural granulosa cell-oocyte uncoupling and the absence of theca development unveil the incomplete efficiency of the system. The present study contributes to explain, from a morphological point of view, the effects of culture conditions on the development of mouse in vitro grown follicles and to highlight the necessity of maintaining efficient intercellular communications to obtain large numbers of fully-grown mature germ cells.

Ovarian Toxicity: From Environmental Exposure to Chemotherapy

Unlike men, who have continuous spermatogenesis throughout most of their lifetime, women are born with a fixed supply of follicles, and this number progressively declines with age until the menopause. Beside age, the speed of follicle depletion can be regulated by genetic, hormonal and environmental influences. In the course of their lives, women are exposed to multiple chemicals and radiation sources that can increase the chance of developing permanent infertility and premature ovarian failure (POF). A wealth of experimental data indicate that iatrogenic (chemotherapy, radiotherapy) and xenobiotic agents (e.g., chemicals, pharmaceuticals) are potent ovotoxicants capable of accelerating ovarian reserve depletion. In the present review we reported the negative effects exerted on mammalian ovary by some widely diffused environmental chemicals, as polycyclic aromatic hydrocarbons (PAHs) and dithiocarbamate mancozeb, and by 1-3 butadiene and 4-vinylcycloexene, two occupational chemicals known to be capable of inducing ovarian cancer and infertility. Furthermore, attention has been devoted to the consequences of chemo- and radiotherapy on the ovary, both known to affect reproductive lifespan. Our increasing understanding of metabolic alterations induced by these agents is fundamental to individuate new therapeutic strategies aimed to prevent ovarian dysfunction in fertile women.