281 research outputs found
The cloning of hBok, Bcl2-L12 and ADAMTS-16 and the functional research into their regulation in physiology of the ovary and other reproductive tissues
No full textOvarian folliculogenesis is a complex process involving the development of a considerable
number of small primordial follicles entering a predefined growth pattern finally resulting in
the selection of a single, large preovulatory follicle. This complex process encompasses
either the death or the successful ovulation of ovarian follicles, involving a multitude of
hormonal signalling, proliferation of tissue and cellular apoptosis. Female mammals are
endowed at birth with a finite number of primordial follicles, each consisting of an oocyte
and a single layer of process of follicular degeneration is called atresia, which has been
recognized at the cellular level as apoptosis. It is the ultimate fate of 99.9 % of all 266.000
to 2.000.000 originally available follicles in the ovary to undergo apoptosis. During the
years between menarche and menopause only 400 of this entire population of ovarian
follicles will achieve ovulation.
During the menstrual cycle until ovulation are well characterized and predominantly
regulated by hormones, most notably those secreted by the pituitary. Conversely, a large
proportion of the local, intraovarians mechanisms involved in this selection process are
largely unknown. It is thought that some of the regulatory mechanisms within the ovarian
follicle are modulated by oocyte-centered signalling.
The present series of studies aimed at characterizing the regulation of some novel factors
involved in human ovarian folliculogenesis, most notably the Bcl-2 family member proteins,
together with specific proteases and endocrine mediators thought to be important regulators
of ECM modelling both during ovarian development and ovulation.
The mechanisms involved in atresia in the ovary are still largely unknown, although they are
based on cellular apoptosis and are often hormonally regulated. Dysregulation of apoptosis
in the ovarian follicle may be responsible for female infertility or associated endocrine
disorders. Furthermore, the study of ovarian follicular apoptosis may give clues to the
pathogenesis of related diseases such as ovarian or breast cancer.
From EST of NCBI GenBank derived from non-normalized human ovarian cDNA libraries,
such as the Stanford microarray database, with the TBLASTN program by searching the
GenBank and EST database, we have cloned and identified two novel human Bcl-2-related
genes, the hBok gene (Bcl2-related ovarian killer or Bcl2-L9 gene) and the Bcl2-L12 gene.
We were able to define the function of their gene products and to elucidate their roles both
in folliculogenesis and in cancer. To further explore the functions of these two genes we
cloned 7 Hbok deletion mutants and 5 Bcl2-L12 deletion mutants according to their
structural domains.
In addition, during search of the ovary-specific gene expression database and trying to find
out novel markers of granulosa cell function, we identified a new member of the family of
proteases ADAMTS-16 (a disintegrin-like and metalloproteinase with thrombospondin type
I motifs): This group of proteases is thought to be important in ECM remodelling during
ovarian development and ovulation. We also identified a splicing variant of ADAMTS-16,
which was termed ADAMTS-16s.
The following three conclusions can be drawn from our research work:
a) The hBok protein contains all four Bcl-2 like domains (BH1, 2, 3 and 4) and is a proapoptotic
Bcl-2 protein identified in the ovary. By fluorescence in situ hybridization
(FISH) and in silico analysis, hBok was found to be located on chromosome 2q37.3.
Its expression was detected in various organs and in several hormonally regulated
cancer cells. Expression of hBok was shown to be upregulated in estrogen-dependent
breast cancer by estrogen deprivation and in myocardial cells during hypoxy. Confocal
laser scanning microscopy examinations and subcellular fractionation studies showed
that hBok was distributed both in cytosol and in membrane in healthy cells. Upon
overexpression of hBok or stimulation of apoptosis, hBok became mainly associated
with the intracellular membrane. Furthermore oligomerization were promoted by
BH3-only proteins, such as Bid, Bnip3 and p53 but prevented by BFL-1. hBok was
found to interact with Bnip3. Our findings suggest that functional BH3-only proteins
facilite the oligomerization and insertion of the hBok into the membrane.
b) We cloned another apoptotic-related gene which is found highly expressed in breast,
placenta, and known as Bcl-2 related proline-rich protein - Bcl2-L12. We found that is
localized in the nucleus and that it posses an anti-apoptotic function. As evidenced by
its structure. It can increase in the S/G2 phase of the cell cycle and enhance DNA
replication. We analysed the Bcl2-L12 gene by cloning 5 different deletion mutations
according to its structure domain, including the full length form (828bp), the deleted
N-terminal (BH2-only), the deleted one coiled-coil domain (12cc) which contained 5
special proline-rich motifs (PXXP) without the BH2 and (PPPP) domains. The
transfection of different deleted mutants into cells showed that the Bcl2-L12 deleted
BH2 domain possesses signalling activity outside the nucleus, indicating that believed
the BH2 domain has a functional role for the nuclear localization of Bcl2-L12. Cell
cycle analysis from FACS for Bcl2-L12 and its deletion mutants further confirmed
that Bcl2-L12 has an anti-apoptotic effect in the MG 132 induced cell apoptosis but
not in the STS induced apoptotic pathway. Each of the 5 mutants has their own
function in modifying the cell cycle. Further results showed that the nuclear signalling
would migrate out off the nucleus and and become fixed in the nuclear membrane or
in mitochondria or in other organelles. We confirmed that Bcl2-L12 is regulated by
many other Bcl2 apoptotic family members and that the cell cycle also changed after
co-transfection with these Bcl2 family genes, such as with Bax, Bid, tbid, hBok, Bcl2,
Bcl2XL, Furthermore, we found that Bcl2-L12 is regulated by some circadian rhythm
genes such as clock, Bmall and Rev-erb.
c) We further characterized full length ADAMTS-16, a novel member of the disintegrin
and metalloproteinase with thrombospondin motifs (ADAMTS) family together with
its splicing variant, ADAMTS-16s, the latter containing the metallopeptidase domain
only. ADAMTS-16 is highly expressed in the kidney and in the ovary, where it is
predominantly expressed in luteinizing granulosa cells but only little in cumulus
oophorus. Expression in several cancer tissues was also detected. In fully
differentiated luteinizing granulosa cells, FSH and forskolin induced expression of
ADAMTS-16, suggesting that it is regulated via cAMP pathway. LH did not have an
effect on the expression of ADAMTS-16. ADAMTS-16 is capable of cleaving α2-
macroglobulin, a widely used substrate for proteases. These studies provide the first
evidence that ADAMTS-16 is an active protease of α2-macroglobulin, which is
present in high concentrations in mature ovarian follicles and which is known to
participate both in estradiol production and in the formation of the perifollicular
vascularization. The FSH dependency of ADAMTS-16 expression in granulosa cells
and its protease activity on α2-macroglobulin suggest a role of ADAMTS-16 in
modulating the maturation of ovarian follicles during the late stages of their
development
H. F. Mussche, J. Blngen, C. Conophagos, J. De Geyter, R. Paepe, G. Vandenven, D. Deraymaeker, Thorikos 1969. Rapport préliminaire sur la sixième campagne de fouilles
No full textGinouvès René. H. F. Mussche, J. Blngen, C. Conophagos, J. De Geyter, R. Paepe, G. Vandenven, D. Deraymaeker, Thorikos 1969. Rapport préliminaire sur la sixième campagne de fouilles. In: L'antiquité classique, Tome 44, fasc. 1, 1975. pp. 379-380
Pulsatile subcutaneous versus bolus intramuscolar gonadotropin administration after pituitary suppression with a long-acting GnRH analogue: a controlled prospective study.
No full textThe potential advantages of pulsatile s.c. administration instead of daily bolus i.m.administration of human urinary gonadotrophin preparations were tested after the administration of a long-acting gonadotrophin-releasing hormone (GnRH) analogue within a programme for in-vitro fertilization (IVF) and embryo transfer. First, the pharmacokinetic properties of human urinary gonadotrophins were analysed with immunological and biological methods, both during bolus i.m. injections and during pulsatile s.c. administration. Second, a prospective randomized controlled study was performed in 75 patients undergoing IVF/embryo transfer in whom the effects of pulsatile s.c. administration were compared with the effects of single daily bolus i.m. injections of the same gonadotrophin preparation. The results showed that neither method of gonadotrophin administration induced measurable changes in the serum concentration of luteinizing hormone (LH). Both oestradiol and andro-stenedione concentrations were slightly lower during pulsatile s.c. gonadotrophin administration, suggesting that this method of gonadotrophin administration results in less LH occupying the ovarian LH receptors. Pulsatile s.c. gonadotrophin administration resembles a continuous infusion of follicle-stimulating hormone (FSH). Significant fluctuations in the serum concentrations of FSH were observed during single daily bolus i.m. administration of human urinary gonadotrophins, but the pregnancy rate of IVF/embryo transfer per cycle after pulsatile s.c. administration was not significantly better than after the daily bolus i.m. injection of gonadotrophins (42.1 versus 37.2%). It is concluded that pulsatile s.c. administration of gonadotrophins instead of single daily injections does not improve the pregnancy rate in IVF/embryo transfe
Functional and clinical consequences of mutations in the FSH receptor. Mol Cell Endocrinol
No full textThe follicle-stimulating hormone (FSH) is essential for normal gametogenesis. In females FSH is required for ovarian development and follicle maturation whereas in males FSH determines Sertoli cell number and quantitatively and qualitatively normal spermatogenesis. FSH action is mediated by a G-protein coupled receptor expressed solely in granulosa and Sertoli cells. The FSH-receptor (FSHR) gene is localized on chromosome 2 p21 and spans a region of 54 kb. It consists of ten exons; exon one to nine encode the large extracellular domain and the transmembrane domain is comprised of exon ten. Mutations in the FSHR gene could severely affect gametogenesis and result in infertility. Therefore screening programs have been initiated, in which patients with disturbed fertility were searched for mutations in the FSHR gene. Several Finnish families were identified displaying an inherited pattern of ovarian dysgenesis, a disease leading to streaky underdeveloped ovaries and primary amenorrhea. By genetic linkage the locus of the genetic defect was confined to chromosome 2 p21. Analysis of the FSHR gene resulted in the identification of a mutation (Ala189Val) homozygous in all affected females. Functional studies revealed that the mutation affects the proper protein folding and thereby inactivates the receptor. In a male patient hypophysectomized because of a pituitary tumor, who despite undetectable serum gonadotropins had normal semen parameters, we hypothesized an activating mutation of the FSHR. Screening of exon ten of the FSHR gene resulted in the identification of a Asp567Gly transition in the third intracytoplasmatic loop. Functional studies resulted in a 1.5-fold increase in basal cAMP production compared to wild type FSHR, indicating that the heterozygous mutation leads to a ligand-independent constitutive activation of the FSHR. This patient provides an exceptional model of nature defining the role of FSH in human spermatogenesis. Mutations of the FSHR might have differential effects in each gender. For example activating mutations have not been described in women, therefore it is not clear whether the constitutive activity of the receptor could disturb normal follicular development resulting in certain infertility
Stem cells in the ovary
No full textFor decades, scientists have thought that female mammals are born with a lifetime supply of oocytes in the ovary, irreversibly destined to decline after birth. However, in recent years a significant controversy with regard to the potential replenishing effects of cells from the bone marrow and blood on ovarian follicular renewal has been stirred up. Although these claims have been met with harsh skepticism, if they prove to be true, the current understanding of the female reproductive system must be revisited. Although these observations and allusions have been limited to the mouse system only, they have opened new discussions about the potential consequences of bone marrow transplantation and even blood donation to the replenishment of the female genital system in general. Still, these findings have not been replicated in other research laboratories so far and the proof that oogenesis can be renewed after birth from cells originating in the bone marrow is still lacking.
In contrast to the ongoing controversy with regard to the possibility of ongoing renewal of oogenesis in the ovary and the possible existence of adult germ stem cells, the existence of somatic stem cells in the ovary has not been hypothesized for a long time.
The first part of this study has been performed to confirm the presence of pluripotent or multipotent stem cell populations among granulosa cells collected from mature human ovarian follicles. This work includes attempts to promote the growth of GCs over prolonged time periods in vitro. Previous studies have demonstrated that this is not possible with culture media which contain FSH and androgens. We identify the specific markers for mesenchymal stem cells and mature GCs and differentiate luteinizing GCs into other cell types of the mesenchymal lineage.
Graafian ovarian follicles consist of follicular fluid, one single mature oocyte and several hundred thousands of granulosa cells (GC). Until now, luteinizing GCs are considered to be terminally differentiated, destined to undergo death after ovulation. Present concepts of luteal function, endocrine regulation of early pregnancy and the recruitment of new ovarian follicles are all based on the cyclical renewal of the entire population of GC.
The first part of this study has been performed to confirm the presence of pluripotent or multipotent stem cell populations among granulosa cells collected from mature human ovarian follicles. This work includes attempts to promote the growth of GCs over prolonged time periods in vitro. Previous studies have demonstrated that this is not possible with culture media which contain FSH and androgens. We identify the specific markers for mesenchymal stem cells and mature GCs and differentiate luteinizing GCs into other cell types of the mesenchymal lineage.
In the second part we demonstrate a three-dimensional (3D) pellet culture system containing type I collagen, which together with LIF allowed not only the survival and growth of primary human GCs, but supported a significant subpopulation of GCs to maintain their phenotype and functionality for prolonged time periods
Plasma for biomedical decontamination : from plasma-engineered to plasma-active antimicrobial surfaces
No full textModern society is suffering from many infectious microbes. Developing antimicrobial surfaces for biomedical decontamination and sterilization is one of the strategic solutions to mitigate the spread of infectious pathogens. Here, we outline the paradigm of plasmas for biomedical decontamination by presenting approaches of plasma-engineered antimicrobial surfaces and novel plasma-active antimicrobial surfaces. Low-temperature plasma can not only be used as a material fabrication tool for antimicrobial surface engineering but also be used directly for microbial inactivation by specially designed plasma-active surfaces that can effectively destroy microorganisms through exposure to plasma. The role of plasmas in the two different kinds of antimicrobial surfaces is discussed along with their associated advantages and disadvantages. Future research directions, challenges, and opportunities in both plasma-based antimicrobial surfaces are also critically evaluated. This analysis contributes to the development of next-generation antimicrobial surfaces for future bio-safety
Synthesis of antibacterial composite coating containing nanocapsules in an atmospheric pressure plasma
No full textAntibacterial coating is an important strategy preventing bacterial colonization and biofilm formation. One-step synthesis of nanocapsule-containing antibacterial coatings with controlled release of Ag+ ions was achieved in the current work by aerosol-assisted atmospheric pressure plasma deposition. The experimental parameters of deposition including the discharge power, silver nitrate concentration, aerosol flow rate, continuous and pulsed mode of operation were studied in order to analyze their effects on surface morphology and chemical composition of the coating. Formation of nanocapsules embedded in the polymeric coating was observed. A core-shell structure was found for nanocapsule with silver in the core and polymer in the shell. Antibacterial coatings on polyethylene terephthalate film were studied in terms of Ag+ ion release, antibacterial properties against Escherichia coli and Staphylococcus aureus, and cytotoxicity with murine fibroblasts. Two-phase release kinetics of Ag+ ions was observed as initially a short-term burst release followed by a long-term slow release. It was revealed that high antibacterial efficiency of the coatings deposited on polyethylene terephthalate films can be coupled with low cytotoxicity. These biocompatible antibacterial coatings are very promising in different fields including biological applications
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