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

Ovarian 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.