RESUMEN

STUDY QUESTION: What human tissues and cell types express the X-linked reproductive homeobox (RHOX) gene cluster? SUMMARY ANSWER: The RHOX homeobox genes and proteins are selectively expressed in germ cells in both the ovary and testis. WHAT IS KNOWN ALREADY: The RHOX homeobox transcription factors are encoded by an X-linked gene cluster whose members are selectively expressed in the male and female reproductive tract of mice and rats. The Rhox genes have undergone strong selection pressure to rapidly evolve, making it uncertain whether they maintain their reproductive tissue-centric expression pattern in humans, an issue we address in this report. STUDY DESIGN, SIZE, DURATION: We examined the expression of all members of the human RHOX gene cluster in 11 fetal and 8 adult tissues. The focus of our analysis was on fetal testes, where we evaluated 16 different samples from 8 to 20 weeks gestation. We also analyzed fixed sections from fetal testes, adult testes and adult ovaries to determine the cell type-specific expression pattern of the proteins encoded by RHOX genes. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used quantitative reverse transcription-polymerase chain reaction analysis to assay human RHOX gene expression. We generated antisera against RHOX proteins and used them for western blotting, immunohistochemical and immunofluorescence analyses of RHOXF1 and RHOXF2/2B protein expression. MAIN RESULTS AND THE ROLE OF CHANCE: We found that the RHOXF1 and RHOXF2/2B genes are highly expressed in the testis and exhibit low or undetectable expression in most other organs. Using RHOXF1- and RHOXF2/2B-specific antiserum, we found that both RHOXF1 and RHOXF2/2B are primarily expressed in germ cells in the adult testis. Early stage germ cells (spermatogonia and early spermatocytes) express RHOXF2/2B, while later stage germ cells (pachytene spermatocytes and round spermatids) express RHOXF1. Both RHOXF1 and RHOXF2/2B are expressed in prespermatogonia in human fetal testes. Consistent with this, RHOXF1 and RHOXF2/2B mRNA expression increases in the second trimester during fetal testes development when gonocytes differentiate into prespermatogonia. In the human adult ovary, we found that RHOXF1 and RHOXF2/2B are primarily expressed in oocytes. LIMITATIONS, REASONS FOR CAUTION: While the average level of expression of RHOX genes was low or undetectable in all 19 human tissues other than testes, it is still possible that RHOX genes are highly expressed in a small subset of cells in some of these non-testicular tissues. As a case in point, we found that RHOX proteins are highly expressed in oocytes within the human ovary, despite low levels of RHOX mRNA in the whole ovary. WIDER IMPLICATIONS OF THE FINDINGS: The cell type-specific and developmentally regulated expression pattern of the RHOX transcription factors suggests that they perform regulatory functions during human fetal germ cell development, spermatogenesis and oogenesis. Our results also raise the possibility that modulation of RHOX gene levels could correct some cases of human infertility and that their encoded proteins are candidate targets for contraceptive drug design.

Asunto(s)

RESUMEN

Interactions between germ cells and surrounding somatic cells are central to ovarian development as well as later function. Disruption of these interactions arising from abnormalities in either cell type can lead to premature ovarian failure (POF). The forkhead transcription factor FOXL2 is a candidate POF factor, and mutations in the FOXL2 gene are associated with syndromic and non-syndromic ovarian failure. Foxl2-deficient mice display major defects in primordial follicle activation with consequent follicle loss, and earlier roles in gonadal development and sex determination have also been suggested. However, despite its importance no data presently exist on its expression in the developing human ovary. Expression of FOXL2 mRNA was demonstrated in the human fetal ovary between 8 and 19 weeks gestation, thus from soon after sex determination to primordial follicle development. Expression in the ovary was higher after 14 weeks than at earlier gestation weeks and was very low in the fetal testis at all ages examined. Immunolocalization revealed FOXL2 expression to be confined to somatic cells, both adjacent to germ cells and those located in the developing ovarian stroma. These cells are the site of action of oocyte-derived activin signalling, but in vitro treatment of human fetal ovaries with activin failed to reveal any regulation of FOXL2 transcription by this pathway. In summary, the expression of FOXL2 in somatic cells of the developing human ovary before and during follicle formation supports a conserved and continuing role for this factor in somatic/germ cell interactions from the earliest stages of human ovarian development.

Asunto(s)

RESUMEN

The close relationship between cumulus cell function and oocyte developmental competence indicates that analysis of cumulus gene expression is a potential non-invasive method to aid embryo selection and IVF outcome. Cumulus was isolated from 674 oocytes from 75 women undergoing ICSI and gene expression analysed by quantitative RT-PCR. Cumulus expression of cyclooxygenase 2 (PTGS2) was higher with mature oocytes, whereas brain-derived neurotrophic factor (BDNF) was lower when fertilisation was normal. Expression levels of gremlin (GREM1) and BDNF were weak positive and negative predictors of embryo quality respectively. Ranking of GREM1 expression within cohorts of oocytes showed that oocytes associated with the highest GREM1 expression were more likely to be transferred or cryopreserved than discarded (49 vs 33%, P<0.02), although the clinical pregnancy rate was not significantly different. This study demonstrates both the feasibility and difficulties of this method of analysis in the largest such group studied thus far. Novel relationships between BDNF expression and fertilisation were identified, and the potential value of GREM1 expression as a marker of embryo quality supports the further assessment of GREM1 analysis in the context of embryo selection.

Asunto(s)

RESUMEN

CONTEXT: The regulation of germ cell proliferation and loss during human ovarian development is poorly understood. This is of particular interest at the time leading up to the formation of primordial follicles, at 18 wk gestation onward. OBJECTIVE: The objective of the study was to identify and quantify germ cell proliferation and apoptosis and expression of caspases in the human fetal ovary. DESIGN: This study was a laboratory investigation. SETTING: The study was conducted at a research institute. METHODS: Cell proliferation and apoptosis were detected using immunohistochemical localization of phosphorylated histone H3 and cleaved caspase-3, respectively. Caspases were also detected by immunoblotting. RESULTS: The overall proportion of germ cells in mitosis remained constant between 14 and 19 wk but showed increasing clustering. Caspase-2, -3, -7, -8, and -9 were detected by immunoblotting. There was a significant increase in germ cell apoptosis. A specimen of 20 wk gestation showed similar phosphorylated histone H3 but markedly lower cleaved caspase-3 expression than earlier gestations. Cleaved caspase-3 was not expressed in oocytes that had formed primordial follicles. CONCLUSIONS: These results indicate that as primordial follicle formation is initiated and progresses, there is an increase in both mitotic activity and apoptosis of those germ cells that have not reached the apparently protective environment of the primordial follicle.

Asunto(s)

RESUMEN

The formation of the essential functional unit of the ovary, the primordial follicle, occurs during fetal life in humans. Factors regulating oogonial proliferation and interaction with somatic cells before primordial follicle formation are largely unknown. We have investigated the expression, localisation and functional effects of activin and its receptors in the human fetal ovary at 14-21 weeks gestation. Expression of mRNA for the activin betaA and betaB subunits and the activin receptors ActRIIA and ActRIIB was demonstrated by RT-PCR. Expression of betaA mRNA increased 2-fold across the gestational range examined. Activin subunits and receptors were localised by immunohistochemistry. The betaA subunit was expressed by oogonia, and the betaB subunit and activin receptors were expressed by both oogonia and somatic cells. BetaA expression was increased in larger oogonia at later gestations, but was low in oocytes within newly formed primordial follicles. Treatment of ovary fragments with activin A in vitro increased both the number of oogonia present and oogonial proliferation, as detected by bromodeoxyuridine (BrdU) incorporation. These data indicate that activin may be involved in the autocrine and paracrine regulation of germ cell proliferation in the human ovary during the crucial period of development leading up to primordial follicle formation.

Asunto(s)

RESUMEN

The regulation of germ cell number in the developing ovary is central to female reproduction. Members of the Bcl-2 family of proapoptotic and antiapoptotic proteins have been implicated in this process in rodents. We investigated the expression of Mcl-1, Bcl-2, Bax, and BAD at 13-21 gestational wk in the human fetal ovary and of Mcl-1 in the adult ovary. mRNA expression of Mcl-1 and its short form Mcl-1s, Bcl-2, Bax, and BAD was demonstrated in fetal ovary by RT-PCR. Hybridization array analysis suggested a selective increase in Mcl-1 expression between 14 and 18 wk gestation, which was confirmed by quantitative PCR. There was a corresponding change in the expression of Mcl-1 protein, detected by immunohistochemistry, from germ cells at the periphery of the ovary at 14-16 wk to the largest germ cells, including oocytes within newly formed primordial follicles, at 21 wk. Mcl-1 was also expressed by oocytes of primordial and preantral follicles in the adult. Bax and BAD immunostaining was detected in both somatic and germ cells in the fetal ovary, whereas Bcl-2 was restricted to somatic cells: no changes in expression were observed. Apoptotic cells, detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, were observed in all fetal ovaries but were infrequent. These results confirm that Bcl-2 family members are differentially expressed in several cell types within the developing human ovary. Increased mRNA expression and the changing distribution of Mcl-1 in germ cells as they develop into primordial follicles as well as persistence in the growing oocyte in the adult may indicate an important role for this survival/antiapoptotic factor throughout germ cell development and maturation.

Asunto(s)

RESUMEN

A linear mammalian artificial chromosome vector will require at least three functional elements: a centromere, two telomeres and replication origins. One route to generate such a vector is by the fragmentation of an existing chromosome. We have previously described the use of cloned telomeric DNA to generate and stably rescue truncated derivatives of a human X chromosome in a somatic cell hybrid. Further rounds of telomere-associated chromosome fragmentation have now been used to engineer a human X-derived minichromosome. This minichromosome is estimated to be < 10 Mb in size. In situ hybridization and molecular analysis reveal that the minichromosome has a linear structure, with two introduced telomere constructs flanking a 2.5 Mb alpha-satellite array. The highly truncated chromosome also retains some chromosome-specific DNA, originating from Xp11.21. There is no significant change in the mitotic stability of the minichromosome as compared with the X chromosome from which it was derived.

Asunto(s)

RESUMEN

Pericentric heterochromatin and telomeres have been shown to be capable of repressing the expression of genes located in close proximity. The effect of adjacent structural sequences on gene expression will be important in the design of mammalian artificial chromosomes. In the process of using telomere-containing constructs to generate a deletion panel of the long arm of the human X chromosome, several cell lines were produced which appeared by in situ hybridization to be broken in Xq at or near the centromere. After analysis of end clones rescued from these cell lines, only two produced data consistent with breaks in the alpha satellite array without accompanying rearrangements. The mitotic stability of an X chromosome, with at least 750 kb of the alpha satellite array deleted, was compared to controls where the alpha satellite array remained intact. No significant change in the stability of the chromosome was observed, suggesting that the truncated chromosome has a fully functional mitotic centromere. There was no detectable change in the expression of the hygromycin resistance gene, which is located between a functional centromere and telomere, in this cell line. This study indicates that structural elements flanking a mammalian selectable marker do not result in silencing.

Asunto(s)

RESUMEN

Five monoclonal antibodies (mAb) were raised that bound to the surface of procyclic stage Trypanosoma congolense with high intensity in immunofluorescence. Immunoblot analysis of trypanosome lysates using 3 of these mAb revealed a diffuse SDS-PAGE band of 36-40 kDa. The purified antigen did not react with Coomassie Blue or silver stains, but did stain blue with Stains-all, indicating acidity. For the one mAb tested, the epitope was periodate-sensitive and therefore probably glycan. Although this antigen shares properties with procyclin/PARP, which forms a surface coat on procyclic Trypanosoma brucei, a search in T. congolense for homologues of a procyclin/PARP gene revealed only non-coding sequence of partial similarity. Using a differential screen, a procyclic stage T. congolense cDNA clone was isolated that encoded a putative 256-amino acid protein containing 2 peptides chemically sequenced independently by Beecroft et al. [36]. The protein, termed glutamate and alanine-rich protein (GARP), has potential hydrophobic leader and tail sequences (the latter with potential for replacement by a glycosyl phosphoinositol anchor) and no potential N-linked glycosylation sites. It has no significant sequence homology with known proteins. Antibodies against a translational fusion of GARP bound specifically in Western blots to a band very similar to that detected by the mAb and also to the purified antigen. Immunogold electron microscopy revealed a dense packing of the antigen on the cell surface. It appears that procyclic T. brucei and T. congolense have major surface proteins with structural analogy, but with no sequence homology.

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)