F. Cillo

Association between human oocyte developmental competence and expression levels of some cumulus genes

At present, oocyte selection is mainly based upon morphological criteria but it is generally acknowledged that its reliability requires further improvement. The aim of this study was to determine whether transcript levels in cumulus cells can provide a useful marker of oocyte developmental competence in vitro. A retrospective study was performed on cumulus cells isolated from 90 oocytes retrieved from 45 patients. Upon fertilization, 35 oocytes originated good-quality embryos and 36 developed into poor-quality embryos, whereas 19 failed to be fertilized. Semi-quantitative measurement of hyaluronic acid synthase 2 (HAS2), gremlin1 (GREM1), and pentraxin 3 (PTX3) mRNAs was performed and data for all genes were obtained from all the samples. Cumulus cells isolated from oocytes that originated high-quality embryos on day 3 of culture had HAS2 and GREM1 transcript levels higher than those detected in cells from oocytes that did not fertilize or developed into poor-quality embryos. No differences were observed in PTX3 levels. Results indicate that the measurement of HAS2 and GREM1 levels in cumulus cells would reliably complement the morphological evaluation providing a useful tool for selecting oocytes with greater chances to be fertilized and develop in vitro.

Changes in poly(A) tail length of maternal transcripts during in vitro maturation of bovine oocytes and their relation with developmental competence

Molecules of mRNA are stored in the oocyte cytoplasm in order to be used during the initial phases of embryonic development. The storage takes place during oocyte growth and the extent of poly(A) tail at the 3′ end of the transcripts has emerged as an important regulatory element for determining their stability. The objective of the present study was to analyse changes in polyadenylation levels of mRNA transcripts, stored in bovine oocytes, during in vitro maturation and their possible relation with developmental competence. Oocyte developmental competence was predicted on the basis of the morphological appearance of their originating ovary as previously established (Gandolfi et al. 1997a. Theriogenology 48:1153–1160) and were divided into groups H (high competence) and L (low competence). The length of the poly(A) tail of the following genes, β‐actin (β‐Act), connexin 43, glucose transporter type 1, heat shock protein 70, oct‐4, plakophilin, pyruvate dehydrogenase phosphatase (PDP), and RNA poly(A) polymerase, was determined at the germinal vesicle (GV) and metaphase II (MII) stage. The results indicated that the poly(A) tail of all genes except for β‐Act and PDP, is shorter after in vitro maturation (IVM) in both groups. Moreover, group L oocytes showed a shorter poly(A) tail than group H oocytes in all genes except for β‐Act and PDP, both at GV and MII stage. We conclude that most of the examined transcripts follow the default deadenylation pattern described during oocyte maturation in other species and that a shorter poly(A) tail is correlated with low developmental competence. Mol. Reprod. Dev. 52:427–433, 1999.

EFFECTS OF ENDOCRINE DISRUPTORS ON DEVELOPMENTAL AND REPRODUCTIVE FUNCTIONS

Endocrine disruptors (EDs) are exogenous environmental molecules that may affect the synthesis, secretion, transport, metabolism, binding, action, and catabolism of natural hormones in the body. EDs may thus interact with the endocrine system of animals and humans and can exert this effect even when present in minute amounts. EDs have adverse impacts on a number of developmental functions in wildlife and humans. Critical periods of urogenital tract and nervous system development in-utero and during early post-natal life are especially sensitive to hormonal disruption. Furthermore a wide range of hormone-dependent organs (pituitary gland, hypothalamus, reproductive tract) are targets of EDs disrupting effects in adult subjects, possibly resulting in cell transformation and cancer. At present about 60 chemicals have been identified and characterized as EDs and belong to three main groups: (a) synthetic compounds utilized in industry, agriculture and consumer products; (b) synthetic molecules used as pharmaceutical drugs and (c) natural chemicals found in human and animal food (phytoestrogens). In the present review we will give special attention to the family of Polychlorinated biphenyls (also indicated as PCBs) because of their persistence in the environment, ability to concentrate up the food chain, continued detection in environmental matrices, and ability to be stored in the adipose tissue of animals as well as humans. The detrimental effects of these compounds, and of EDs more in general, on health and reproduction will be discussed, presenting experimental data aimed at understanding the molecular mechanisms involved in their action.

Somatostatin up-regulates topoisomerase II alpha expression and affects LNCaP cell cycle

mRNA differential display-PCR analysis was used to perform a systematic screening of Somatostatin (SS)-regulated genes in the human prostatic carcinoma cell line LNCaP (Lymph Node Carcinoma of the Prostate). A 170 bp fragment was shown to be up-regulated by SS. Sequence analysis of this fragment revealed its homology with the human Topoisomerase II Alpha gene. Up-regulation of Topoisomerase II Alpha was confirmed by Northern blot hybridisation and was induced by the same dose of SS (1 nM) earlier demonstrated to inhibit LNCaP cell growth. Furthermore, SS possible effects on timing, as well as concentration of Topoisomerase II Alpha along the different phases of the cell cycle were investigated. To this purpose changes in the enzyme protein concentration in response to SS were assessed in synchronised LNCaP cells. The hormone was shown to exert a perturbing effect on both parameters considered, possibly related to its inhibitory action on LNCaP cell replication.

Oocytes and Ovarian Follicles As Targets Of Endocrine Disrupters: Consequences For Reproductive Health

This chapter illustrates the physiological characteristics of oocytes and ovarian follicles in relation with their possible exposure to environmental contaminants. The total amount of oocytes present in the adult ovary is established shortly before birth. As soon as the primordial follicle store is established, follicle recruitment begins and it continues without halting for the rest of life or until the ovary is depleted. Between recruitment and ovulation, the oocyte goes through a deep transformation in order to become able to sustain embryonic development. The permanent nature of the oocyte population together with its essential role in supporting embryonic development makes it a sensitive target for the adverse effects of environmental contaminants. Amongst the different environmental contaminants we focussed our analysis on a range of chemicals known as endocrine disrupters (EDs) because of their widespread diffusion and the potential hazard they represent for reproductive health. Their sites of action include the hypothalamus-hypophyseal system, resulting in disruption of the normal pattern of gonadotropin secretion, and the ovary, resulting in destruction of the oocyte. In turn, oocyte destruction can result from directly impairing oocyte viability or from indirect mechanisms involving alterations within the follicular wall. Recent work performed in our laboratory on bovine and pig, has begun to elucidate some of the cellular and molecular mechanisms involved in EDs negative effects on oocyte developmental competence. Our results indicate that EDs perturb maternal mRNA stability and disrupt the physiological remodelling of the cytoplasm, taking place during oocyte maturation.

257 EXPRESSION PROFILING OF GENES CRUCIAL FOR LINEAGE DETERMINATION IN IN VITRO-DERIVED EARLY BOVINE EMBRYOS

In the early blastocyst, lineage segregation depends on the expression of several key specific transcription factors. In the mouse, commitment to inner cell mass (ICM), lineage is positively regulated by Oct-4, a repressor of trophectoderm (TE) cell fate, and Nanog, which inhibits the formation of extra-embryonic and primitive endoderm. Cdx2, a caudal-type homeodomain protein, is specifically expressed in the nascent TE. The mechanisms that drive Cdx2 segregation to the outside cells are still unclear. However, the expression of Fgf Receptor 2 (FgfR2), restricted to the outside cells, and the role for its ligand, Fgf4, in promoting TE development, suggest that this signalling pathway may act upstream or in parallel with Cdx2. Little information is available on these genes in bovine; therefore the aims of the present study were as follows: (a) to identify and characterize the expression profiles of Cdx2 and FgfR2 variants (IIIc and IIIb) in bovine oocytes and pre-implantation embryos; and (b) to compare their expression patterns in ICM and TE with that of Oct-4 and Nanog. Bovine oocytes and embryos were obtained by in vitro maturation and fertilization; blastocysts at Day 7 post-insemination underwent microsurgery to separate TE from ICM. RNA was isolated from MII oocytes; 2-, 4-, 8-, and 16-cell embryos; morulae; blastocysts; ICMs; and TEs. Semi-quantitative analysis of Cdx2 and FgfR2 expression in oocytes and embryos was performed in the exponential phase of PCR amplification with rabbit globin as exogenous control. In order to exclude false negative results, PCR amplification in isolated TE and ICM was extended to the plateau phase for all genes considered. Fragment identity was confirmed by sequencing. Comparison of bovine Cdx2 cDNA sequence (EMBL AM293662) with databases revealed a 91% and 87% homology with human and mouse, respectively. Cdx2 expression was not detectable in MII oocytes, but increased in 2-cell embryos. Transcript levels decreased at the 4- and 8-cell stages and then increased again in the blastocyst. FgfR2 variants were present as both maternal and embryonic transcripts, because they were detectable throughout pre-implantation development. Cdx2 and FgfR2 IIIc and IIIb expression was restricted to TE cells. Nanog was detected only in ICM, whereas Oct-4 was expressed in both lineages, as previously described in bovine (van Eijk et al. 1999 Bio. Reprod. 60, 1093-1103). In conclusion, the expression profiles of Nanog, Cdx2, and FgfR2 in bovine pre-implantation embryos follow the pattern previously described in the mouse. Their differentially segregated expression is consistent with their role as selector factors of ICM vs. TE fates. The significance of Oct-4 ubiquitous distribution still remains to be elucidated. This work was supported by FIRB RBNE01HPMX_005, TECLA-MIUR, and EUROSTELLS-ESF.