Marina Prisco

Slow cooling of human oocytes: ultrastructural injuries and apoptotic status

OBJECTIVE To identify the damages caused by slow cooling human metaphase II (MII) oocytes comparing the ultrastructure, inner mitochondrial membrane potential (DeltaPsim), and apoptotic status of fresh and cryopreserved oocytes. DESIGN Experimental study. SETTING University biology research unit and private IVF unit. PATIENT(S) Fresh and cryopreserved supernumerary MII oocytes donated from women undergoing IVF cycles. MAIN OUTCOME MEASURE(S) Ultrastructure was assessed by transmission electron microscopy (TEM), mitochondrial function by means of the fluorescent DeltaPsim reporter JC-1, and apoptotic status through fluorescent labeling with the pan-caspase inhibitor fluorescein isothiocyanate conjugate (FITC)-VAD FMK, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. RESULT(S) Compared to fresh oocytes, frozen/thawed (F/T) oocytes showed reduced cortical granule densities (F/T 3.35 +/- 1.94/10 microm vs. fresh 10.30 +/- 3.9/10 microm), swelling of smooth endoplasmic reticulum (F/T 0.084 +/- 0.03 microm(2) vs. fresh 0.040 +/- 0.02 microm(2)), decreased electron density of the mitochondrial matrix and damage to the mitochondrial membranes, low DeltaPsim of pericortical mitochondria, but no signs of apoptosis. CONCLUSION(S) Slow cooling is associated with cortical granule exocytosis, swelling of smooth endoplasmic reticulum vesicles, and mitochondrial damage, but does not induce early or late apoptotic events. The observed injuries might be responsible for the reduced developmental competence of cryopreserved oocytes.

An ultrastructural study on the vitellogenesis in the spotted ray Torpedo marmorata

The present investigation strongly suggests that in Torpedo the oocyte growth is not only due to the uptake of exogenous molecules, but also by the oocyte itself and the granulosa cells. The oocyte, starting from the early previtellogenic follicles (see also Mol. Reprod. Dev. 61 (2002) 78), synthesizes large amounts of glycogen. Later, as the oocyte growth goes on, the cytoplasm of granulosa cells progressively bears numerous islets of glycogen, which are also evident inside the intercellular bridges and in the oocyte cortex, suggesting that they may flow from granulosa cells to the oocyte. The contribution of granulosa cells seems to become most relevant during the vitellogenesis. In vitellogenic follicles, both small, intermediate, and pyriform-like cells bear numerous vacuoles containing vitellogenin-like material, suggesting strongly that in Torpedo, differently from other vertebrate species, granulosa cells could be engaged in vitellogenesis. The present investigation does not allow us to know if such a material is due to a transcytosis process and/or is synthesized inside them. The organization of granulosa seems to exclude the possibility that it is transferred to granulosa via transcytosis. On the contrary, granulosa cells, especially in vitellogenic follicles, display the morphological organization of metabolically active cells, so they could be engaged in vitellogenin synthesis. This interpretation is consistent with the observation that granulosa cells are positively stained by OZI (osmium tetroxide-zinc iodide) and that the same positivity is evident on intercellular spaces, containing vitellogenin-like material, and on nascent yolk globules.

An ultrastructural study of germ cells during ovarian differentiation in Torpedo marmorata

An ultrastructural investigation, performed on embryos, neonates, subadult and adult females, demonstrated that in Torpedo marmorata oogenesis occurs very early in life and continues, in its proliferative phase, also after birth. Clusters of early meiotic cells were already evident in the ovarian cortex of 6‐cm‐long embryos, as well as in the ovary of newborns and three‐month‐old young. Conversely, in the ovaries of subadult and adult females, all the germ cells present were organized into follicles, and no clusters of oogonia and early meiotic cells were generally found in the cortex, except for one adult female where clusters of germ cells not organized in follicles were found in the cortex. These data demonstrated that, in Torpedo marmorata, oogenesis is immediate, and, as oogonia persist after birth, more similar to that of mouse, monkey, rabbit, and ferret (Mauleon Arch Anat Microsc, 1967; 56:125–150; Byskov and Hoyer 1994 ) than to that of human, rat, pig, and guinea pig (Byskov and Hoyer 1994 ). Such a pattern is in agreement with the reproductive strategy of Torpedo, a scantly prolific species with low uterine fecundity. The presence of meiotic cells that are not organized in follicles in one adult female might be consistent with the large individual variability characterizing cartilaginous fishes. The possibility that such a character is typical of mature females should be rejected as oogonia and early meiotic cells were not found inside the totally sectioned gonads of subadult and adult females. Anat Rec 263:237–245, 2001.

Intercellular bridges between granulosa cells and the oocyte in the elasmobranch Raya asterias

In the present ultrastructural study intercellular bridges, connecting somatic granulosa cells to oocyte, have been detected for the first time and their modifications have been followed during Raja oogenesis. Intercellular bridges make their first appearance in small previtellogenic follicles as connecting devices between small cells and the oocyte. Later on, when the follicular epithelium becomes polymorphic and multilayered, for the presence of small, large, and pyriform‐like cells, intercellular bridges link the oocyte and the different granulosa cells. Intercellular bridges contain ribosomes, whorl of membranes, mitochondria and vacuoles. Such cytoplasmic components are present also in the cell apex of large and pyriform‐like cells thus suggesting, in agreement with other species (Motta et al. J. Exp. Zool., 1996;276:223–241) they may flow toward the oocyte. In this regard the presence of intercellular bridges during the oogenesis of cartilagineous fish may represent a crucial event of the active cooperation between granulosa cells and the oocyte. Anat Rec 255:180–187, 1999.

Chronic Pro-oxidative State and Mitochondrial Dysfunctions are more Pronounced in Fibroblasts from Down Syndrome Foeti with Congenital Heart Defects

Trisomy of chromosome 21 is associated to congenital heart defects in ∼50% of affected newborns. Transcriptome analysis of hearts from trisomic human foeti demonstrated that genes involved in mitochondrial function are globally downregulated with respect to controls, suggesting an impairment of mitochondrial function. We investigated here the properties of mitochondria in fibroblasts from trisomic foeti with and without cardiac defects. Together with the upregulation of Hsa21 genes and the downregulation of nuclear encoded mitochondrial genes, an abnormal mitochondrial cristae morphology was observed in trisomic samples. Furthermore, impairment of mitochondrial respiratory activity, specific inhibition of complex I, enhanced reactive oxygen species production and increased levels of intra-mitochondrial calcium were demonstrated. Seemingly, mitochondrial dysfunction was more severe in fibroblasts from cardiopathic trisomic foeti that presented a more pronounced pro-oxidative state. The data suggest that an altered bioenergetic background in trisomy 21 foeti might be among the factors responsible for a more severe phenotype. Since the mitochondrial functional alterations might be rescued following pharmacological treatments, these results are of interest in the light of potential therapeutic interventions.

A network system for vitellogenin synthesis in the mussel Mytilus galloprovincialis (L.)

The aim of this study is to assess, by RT‐PCR, in situ hybridization, electron microscopy, and immunohistochemistry, the site/s of vitellogenin (VTG) synthesis in the mussel Mytilus galloprovincialis. Our investigations demonstrate that, among the analyzed tissues, the synthesis of VTG occurs only in the female gonad, that is, within the oocyte and follicle and connective cells. Such a synthesis is just evident in early vitellogenic oocytes, whose cytoplasm is characterized by numerous RER cisternae and an extended Golgi complex surrounded by nascent yolk platelets. The synthesis of VTG goes on in vitellogenic oocytes assuming a pear form, and progressively reduces once the oocyte shows the pear or polygonal form, typical of those oocytes that have concluded the growth. The expression of VTG occurs also within follicle (auxiliary) and connective cells. In particular, it is noteworthy that follicle cells are characterized by numerous RER cisternae and an active Golgi complex surrounded by numerous vesicles and vacuoles containing electron dense material. The same material is also present along their plasma membrane, within the intercellular space between oocyte and follicle cells, and finally within invaginations of the oocyte surface, thus suggesting a VTG transfer to the oocyte via endocytosis. Differently, no VTG synthesis was observed within digestive gland. All together the findings here reported strongly suggest that in M. galloprovincialis, inside the gonad, the VTG synthesis occurs in the oocyte (autosynthesis) and in the follicle and adipogranular cells (heterosynthesis). J. Cell. Physiol. 228: 547–555, 2013.

Molecular characterization and gene expression of the pituitary adenylate cyclase-activating polypeptide (PACAP) in the lizard brain.

The pituitary adenylate cyclase-activating polypeptide (PACAP) is considered a pleiotropic neuropeptide in vertebrate physiology. The nucleotide sequence, the expression and the distribution of PACAP were determined in the brain of the lizard Podarcis sicula. RT-PCR showed that the brain of this reptile synthesizes an mRNA coding for PACAP. By performing in situ hybridization and immunohistochemistry techniques, a wide distribution of PACAP and its mRNA in neurons, nervous fibers and other cells was found. Phylogenetic sequence analysis indicates that lizard PACAP is highly conserved, resembling the vertebrate PACAP. Our data demonstrate that PACAP is not only highly preserved during vertebrate evolution but also suggest that PACAP could be implicated in a wide number of functions in the physiology of the reptile brain.

Experimentally nonylphenol-polluted diet induces the expression of silent genes VTG and ERα in the liver of male lizard Podarcis sicula

Endocrine Disruptor Chemicals (EDCs) with estrogen-like properties i.e nonylphenol (NP) induce vitellogenin (VTG) synthesis in males of aquatic and semi-aquatic species. In the oviparous species VTG is a female-specific oestrogen dependent protein. Males are unable to synthesize VTG except after E2 treatment. This study aimed to verify if NP, administered via food and water, is able to induce the expression of VTG even in males of vertebrates with a terrestrial habitat such as the lizard Podarcis. By means of ICC, ISH, W/B and ELISA we demonstrated that NP induces the presence of VTG in the plasma and its expression in the liver. VTG, undetectable in untreated males, reaches the value of 4.34 μg/μl in the experimental ones. Expression analysis and ISH in the liver showed that an NP-polluted diet also elicits the expression of ERα in the liver which is known to be related to VTG synthesis in Podarcis.

Effects of nonylphenol on vitellogenin synthesis in adult males of the spotted ray Torpedo marmorata

The aim of this investigation was to assess the effects of nonylphenol (NP), an oestrogen-like environmental pollutant, on the vitellogenin (VTG) synthesis in adult males of the aplacental viviparous cartilaginous fish Torpedo marmorata. The VTG recovery in males is considered a biomarker of xeno-oestrogenic pollution as this lipophosphoglycoprotein is physiologically induced by oestrogens only in females of oviparous and ovoviparous vertebrates. Using in situ hybridization and immunohistochemistry, T. marmorata males injected with nonylphenol showed the presence of VTG in the liver and the kidney. In particular, vtg messenger (m)RNA and VTG protein were expressed in the liver, whereas in the kidney cells only the presence of VTG was recorded. By contrast, no expression for VTG was detected in the testis. These results demonstrate that in T. marmorata NP induces the expression of vtg only in the liver; the presence of VTG in the kidney and its absence in the testis are discussed.

Hepatic mitochondrial energetics during catch-up fat after caloric restriction

The objective of the study was to investigate whether changes in liver mitochondrial energetics could underlie the enhanced energetic efficiency that drives accelerated body fat recovery (catch-up fat) during refeeding after caloric restriction. Rats were subjected to caloric restriction (50% of ad libitum intake) for 15 days and then refed for 1 or 2 weeks on an amount of chow equal to that of controls matched for weight at the onset of refeeding. Whole-body metabolism was characterized by energy balance and body composition determinations as well as by indirect calorimetric measurements of 24-hour energy expenditure, substrate oxidation, and whole-body de novo lipogenesis estimated from nonprotein respiratory quotient. Hepatic mitochondrial energetics were determined from measurements of liver mitochondrial mass, respiratory capacities, and proton leak (both basal and fatty acid stimulated), whereas hepatic oxidative status was assessed from measurements of hepatic mitochondrial lipid peroxidation, aconitase, and superoxide dismutase activity. Furthermore, hepatic lipogenic capacity was determined from assays of fatty acid synthase activity. Compared with controls, isocalorically refed rats showed an elevated energetic efficiency and body fat gain over both week 1 and week 2 of refeeding, as well as a lower 24-hour energy expenditure and higher rates of whole-body de novo lipogenesis at the end of both week 1 and week 2 of refeeding. Analysis of the liver revealed that after 1 week (but not after 2 weeks) of refeeding, the mitochondrial mass (but not mitochondrial density) was lower in refed rats than in controls, associated with higher state 3 mitochondrial respiratory capacity, increased superoxide dismutase activity, as well as higher fatty acid synthase activity. These results suggest that, although at the whole-body level elevations in energy efficiency and de novo lipogenesis are coordinated toward catch-up fat, the overall hepatic mitochondrial energetic status during refeeding is more consistent with a contributory role of the liver in the enhanced de novo lipogenic machinery during catch-up fat rather than in the energy-conservation mechanisms (elevated energetic efficiency) that spare energy for catch-up fat.