Giving bodies to ghosts: locating molecules in the very place where they exert their biological roles.

This paper reviews some of the goals of our investigations published over the years on Rivista di Istochimica Normale e Patologica, Basic and Applied Histochemistry, and the European Journal of Histochemistry - EJH. In a series of papers, we published some of the basic cytochemical features of the sperm cytodifferentiation process for the first time. This was a conceptual and practical prerequisite to the in situ quantitative evaluation of sperm DNA content. We showed that the discrepancy between the expected 1:2 ratio when comparing sperm versus somatic cell DNA content (sperm DNA content is always far low from the theoretical value) is due to DNA losses caused by the hydrochloric treatment entailed by the Feulgen reaction. The knowledge of the specific losses that occur during the various steps of the Feulgen reaction has allowed us to use it critically in Genome Size studies to highlight: - sperm aneuploidy in chromosomally derived subfertility; - the broad variability range of Mammalian genome sizes; - that termites are roaches (after decades of discussion on this topic). In addition, in a seminal paper on human oocytes, we showed (by transmission electron microscopy) a specific chromatin and cytoplasmic organization (both essential for further embryo development) linked to oocyte maturation arrest, a datum quite relevant to treating unmet therapeutic needs in human and veterinary reproduction.

Genome size evaluations in cockroaches: new entries.

In this paper, we report genome size (GS) values for nine cockroaches (order Blattodea, families Blattidae, Blaberidae and Ectobiidae, ex Blattelidae,), three of which are original additions to the ten already present in the GS database: the death's head roach (Blaberus craniifer), the Surinam cockroach (Pycnoscelus surinamensis) and the Madeira cockroach (Leucophaea maderae). Regarding the American cockroach (Periplaneta americana), the GS database contains two contrasting values (2.72 vs 3.41 pg); likely, the 2.72 pg value is the correct one as it is strikingly similar to our sperm DNA content evaluation (2.80 ± 0.11 pg). Also, we suggest halving the published GS of the Argentine cockroach Blaptica dubia and the spotted cockroach (the gray cockroach) Nauphoeta cinerea discussing (i) the occurrence of a correlation between increasing 2N chromosome number and GS within the order Blattodea; and (ii) the possible occurrence of a polyploidization phenomenon doubling a basic GS of 0.58 pg of some termite families (superfamily Blattoidea, epifamily Termitoidae).

Sertoli Cells - Methods and Protocols.

The mammalian somatic cell providing the physiological milieu to germ cells growth and differentiation is the Sertoli cell, named after the Italian physiologist Enrico Sertoli. Sertoli described for the first time this functionally multitasking cell in 1865 (when he was 23 years-old!) in human testicular tissues while ending up his medicine studies at the University of Pavia...

Cytoplasmic lattices are not linked to mouse 2-cell embryos developmental arrest.

Cytoplasmic lattices are important regulators of oocyte maturation. They store components of the protein synthesis machinery including ribosomes and, among others, they are involved in the regulation of microtubule dynamics in both mouse and human. Cytoplasmic lattices undergo dramatic reorganizations at crucial stages of oocyte maturation, where they are abundantly present in the cytoplasm of developmentally competent oocytes named SN (Surrounded Nucleolus) while they are rare in the cytoplasm of 2-cell stage-arresting NSN (Not Surrounded Nucleolus) oocytes, suggestive of a requirement of cytoplasmic lattices for development past the 2-cell stage. Here, to elucidate this requirement, 2-cell mouse embryos derived from SN and NSN oocytes were analyzed by transmission electron microscopy. Contrary to what had been proposed hitherto, cytoplasmic lattices are present in 2-cell embryos derived not only from SN, but also from NSN oocytes, irrespective of the embryo production system (intra cytoplasmic sperm injection, parthenogenesis). Hence our conclusion that cytoplasmic lattices do not count among the factor(s) responsible for the embryo arrest at this crucial stage of development.

Next-generation nutritional biomarkers to guide better health care.

One of the main precision medicine's promises is nutrigenomics; still far from being a widespread attitude for the laypeople, it is certainly one of the most challenging aspect of the near to come health care system based on personalized medicine. Thus, we welcome books like this one that is exploring the pre-requisites to get a better health care in the new millennium.....

The egg. The inside story of a cell.

The egg, a fantastic little laboratory of molecular biology, has played a crucial role in redefining modern biology by moving it from the description of living things to the synthesis of living things (synthetic biology). Over the centuries, many hypotheses have been advanced concerning the egg's role in reproduction-from the preformation theory until von Baer's discovery to the present, with the 2012 Nobel Prize for Physiology or Medicine celebrating the egg as a totipotent stem cell able to reprogram fully differentiated somatic nuclei. The molecular dissection of its cytoplasmic components makes the egg an ideal bioreactor for several biotechnological applications, including pharmacological and food production sciences. In addition to its ubiquitous contribution to the worldwide diet, the egg, a powerful symbol, pervades philosophy, art, religion, and idiomatic expressions.

Oogenesis specific genes (Nobox, Oct4, Bmp15, Gdf9, Oogenesin1 and Oogenesin2) are differentially expressed during natural and gonadotropin-induced mouse follicular development.

Using a semi-quantitative, single-cell sensitive RT-PCR method, we studied the expression of oogenesis specific genes (Nobox, Oct4, Bmp15, Gdf9, Oogenesin1 and Oogenesin2) in single oocytes collected from primordial, primary, secondary, preantral and antral follicles during natural and gonadotropin-induced mouse follicular development. We compared the number of transcripts of these genes, showing that they are differentially expressed, both in natural conditions and under gonadotropin-induction throughout the assessed developmental stages. Our data show a clear increase in the number of transcripts between the primordial until the preantral stages, with the exception of the Oogenesin1 transcripts under gonadotropin-induction. The number of transcripts starts decreasing at the antral stage and proceeds until the metaphase II stage, with values very similar to those obtained for the primordial oocytes in both analyzed conditions. Under exogenous gonadotropin-induction, oocyte recruitment to ovulation at the preantral stage is marked by an increase in Nobox and Oogenesin2 gene expression that is concomitant with a decrease in Oogenesin1 gene expression. Oocytes that are able to proceed into whole embryo development show a tight regulation of Nobox and Oct4 expression at the antral stage. A parallel immunocytochemical study at the protein level corroborates these findings.

Gonadotropins affect Oct-4 gene expression during mouse oocyte growth.

The transcription factor Oct-4 is required for the maintenance of stem cells pluripotency and is involved in the regulation of the expression of a number of developmental genes. Oct-4 is also expressed in the female gamete during folliculogenesis, but the role it plays is largely unknown. Its upstream and promoter regions have some characteristic features that make this gene a possible target of hormonal regulation. To further our understanding of Oct-4 gene expression during oocyte growth, we tested whether changes to the hormonal milieu of the ovary may affect its transcription. Using a semi-quantitative single-cell-sensitive reverse transcription-polymerase chain reaction (RT-PCR) assay, we investigated the pattern of Oct-4 expression during mouse oocyte growth in females intraperitoneally injected with pregnant mare serum gonadotropin (PMSG) alone or PMSG followed by human chorionic gonadotropin (hCG). The results of this study show that gonadotropins induced two major increases in Oct-4 expression during folliculogenesis: (1) 48 hr after PMSG injection, in oocytes isolated from primordial follicles; and (2) following a surge of hCG, in preovulatory antral oocytes. These results suggest a potential twofold role for this gene in the recruitment of oocytes for initiating growth and in the selection of oocytes for ovulation. Also, they may contribute to our knowledge of the molecular bases of oocyte growth, meiosis resumption, and acquisition of a developmental competence.