BCL2-modifying factor promotes germ cell loss during murine oogenesis.

Apoptosis plays a prominent role during ovarian development by eliminating large numbers of germ cells from the female germ line. However, the precise mechanisms and regulatory proteins involved in germ cell death are yet to be determined. In this study, we characterised the role of the pro-apoptotic BH3-only protein, BCL2-modifying factor (BMF), in germ cell apoptosis in embryonic and neonatal mouse ovaries. BMF protein was immunohistochemically localised to germ cells at embryonic days 15.5 (E15.5) and E17.5 and postnatal day 1 (PN1), coincident with entry into the meiotic prophase, but was undetectable at E13.5, and only present at low levels at PN3 and PN5. Consistent with this expression pattern, loss of BMF in female mice was associated with a decrease in apoptosis at E15.5 and E17.5. Furthermore, increased numbers of germ cells were found in ovaries from Bmf(-/-) mice compared with WT animals at E15.5 and PN1. However, germ cell numbers were comparable between Bmf(-/-) and WT ovaries at PN3, PN5 and PN10. Collectively, these data indicate that BMF mediates foetal oocyte loss and its action limits the maximal number of germ cells attained in the developing ovary, but does not influence the number of primordial follicles initially established in ovarian reserve.

PUMA regulates germ cell loss and primordial follicle endowment in mice.

The number of primordial follicles initially established within the ovary is influenced by the extent of germ cell death during foetal ovarian development, but the mechanisms that mediate this death have not been fully uncovered. In this study, we identified BBC3 (PUMA) (p53 upregulated modulator of apoptosis, also known as BCL2-binding component 3), a pro-apoptotic BH3-only protein belonging to the BCL2 family, as a critical determinant of the number of germ cells during ovarian development. Targeted disruption of the Bbc3 gene revealed a significant increase in the number of germ cells as early as embryonic day 13.5. The number of germ cells remained elevated in Bbc3(-/-) female mice compared with WT female mice throughout the remainder of embryonic and early postnatal life, resulting in a 1.9-fold increase in the number of primordial follicles in the ovary on postnatal day 10. The increase in the number of germ cells observed in the ovaries of Bbc3(-/-) mice could not be attributed to the altered proliferative activity of germ cells within the ovaries. Furthermore, BBC3 was found to be not required for the massive germ cell loss that occurs during germ cell nest breakdown. Our data indicate that BBC3 is a critical regulator of germ cell death that acts during the migratory phase of oogenesis or very soon after the arrival of germ cells in the gonad and that BBC3-mediated cell death limits the number of primordial follicles established in the initial ovarian reserve.

A single amino acid deletion in the ER Ca2+ sensor STIM1 reverses the in vitro and in vivo effects of the Stormorken syndrome-causing R304W mutation.

Stormorken syndrome is a multiorgan hereditary disease caused by dysfunction of the endoplasmic reticulum (ER) Ca2+ sensor protein STIM1, which forms the Ca2+ release-activated Ca2+ (CRAC) channel together with the plasma membrane channel Orai1. ER Ca2+ store depletion activates STIM1 by releasing the intramolecular "clamp" formed between the coiled coil 1 (CC1) and CC3 domains of the protein, enabling the C terminus to extend and interact with Orai1. The most frequently occurring mutation in patients with Stormorken syndrome is R304W, which destabilizes and extends the STIM1 C terminus independently of ER Ca2+ store depletion, causing constitutive binding to Orai1 and CRAC channel activation. We found that in cis deletion of one amino acid residue, Glu296 (which we called E296del) reversed the pathological effects of R304W. Homozygous Stim1 E296del+R304W mice were viable and phenotypically indistinguishable from wild-type mice. NMR spectroscopy, molecular dynamics simulations, and cellular experiments revealed that although the R304W mutation prevented CC1 from interacting with CC3, the additional deletion of Glu296 opposed this effect by enabling CC1-CC3 binding and restoring the CC domain interactions within STIM1 that are critical for proper CRAC channel function. Our results provide insight into the activation mechanism of STIM1 by clarifying the molecular basis of mutation-elicited protein dysfunction and pathophysiology.

A child with mosaicism for deletion (14)(q11.2q13).

In this case report we describe a child with a de novo deletion in the (q11.2q13) region of chromosome 14. The child presented with dysmorphic features - anophthalmia, microcephaly, and growth retardation. Cytogenetic studies showed mosaicism. The karyotype was 46,XX,del(14)(q11.2;q13) [16] /46,XX [9]. We compared the features observed in this child with that of others with the same deletion reported in scientific literature and found that this is the first report of a child mosaic for this deletion. It is also the first time it has been reported in association with anophthalmia.

STIM1 R304W in mice causes subgingival hair growth and an increased fraction of trabecular bone.

Calcium signaling plays a central role in bone development and homeostasis. Store operated calcium entry (SOCE) is an important calcium influx pathway mediated by calcium release activated calcium (CRAC) channels in the plasma membrane. Stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum calcium sensing protein important for SOCE. We generated a mouse model expressing the STIM1 R304W mutation, causing Stormorken syndrome in humans. Stim1R304W/R304W mice showed perinatal lethality, and the only three animals that survived into adulthood presented with reduced growth, low body weight, and thoracic kyphosis. Radiographs revealed a reduced number of ribs in the Stim1R304W/R304W mice. Microcomputed tomography data revealed decreased cortical bone thickness and increased trabecular bone volume fraction in Stim1R304W/R304W mice, which had thinner and more compact bone compared to wild type mice. The Stim1R304W/+ mice showed an intermediate phenotype. Histological analyses showed that the Stim1R304W/R304W mice had abnormal bone architecture, with markedly increased number of trabeculae and reduced bone marrow cavity. Homozygous mice showed STIM1 positive osteocytes and osteoblasts. These findings highlight the critical role of the gain-of-function (GoF) STIM1 R304W protein in skeletal development and homeostasis in mice. Furthermore, the novel feature of bilateral subgingival hair growth on the lower incisors in the Stim1R304W/R304W mice and 25 % of the heterozygous mice indicate that the GoF STIM1 R304W protein also induces an abnormal epithelial cell fate.

STIM1 R304W causes muscle degeneration and impaired platelet activation in mice.

STIM1 and ORAI1 regulate store-operated Ca2+ entry (SOCE) in most cell types, and mutations in these proteins have deleterious and diverse effects. We established a mouse line expressing the STIM1 R304 W gain-of-function mutation causing Stormorken syndrome to explore effects on organ and cell physiology. While STIM1 R304 W was lethal in the homozygous state, surviving mice presented with reduced growth, skeletal muscle degeneration, and reduced exercise endurance. Variable STIM1 expression levels between tissues directly impacted cellular SOCE capacity. In contrast to patients with Stormorken syndrome, STIM1 was downregulated in fibroblasts from Stim1R304W/R304W mice, which maintained SOCE despite constitutive protein activity. In studies using foetal liver chimeras, STIM1 protein was undetectable in homozygous megakaryocytes and platelets, resulting in impaired platelet activation and absent SOCE. These data indicate that downregulation of STIM1 R304 W effectively opposes the gain-of-function phenotype associated with this mutation, and highlight the importance of STIM1 in skeletal muscle development and integrity.

Localisation of the Notch family in the human endometrium of fertile and infertile women.

To investigate the spatial and temporal immunolocalisation and staining intensity of the Notch signalling family in endometrium of fertile and infertile women, endometrial biopsies were collected by curettage from 25 fertile women across the menstrual cycle and 10 infertile women in the mid secretory phase of menstrual cycle. Immunohisotchemistry was completed for NOTCH1, -2, -3, -4, cleaved Notch, DLL1, -3, -4, JAGGED1, -2, HES and NUMB and immunostaining intensity measured in both the endometrial glandular and luminal epithelium. NOTCH1 and the ligands DLL1 and JAGGED1 were key proteins displaying increased staining intensity during the receptive phase of the menstrual cycle and dysregulated in infertile endometrium. Conversely, NUMB a negative regulator of Notch signalling was decreased in the mid secretory phase of the menstrual cycle in fertile women and increased with infertility.

A balanced de novo inv(7)(p14.3q22.3) disrupting PDE1C and ATXN7L1 in a 14-year old developmentally delayed boy.

We report a 14 year old male patient ascertained for developmental delay, carrying a de novo pericentric inversion on chr(7)(p14.3q22.3). Sequencing revealed that the breakpoints overlap a LTR sequence on 7q22.3 and a LINE on 7p14.3. A TTTAAA motif was found in proximity of the breakpoints on both arms. In addition the sequencing detected several small micro-rearrangements, deletion, duplication, insertion, at the breakpoints. No significant sequence identity exists between the 7p14.3 and 7q22.3 breakpoints. These features at the breakpoint junctions suggest that the inversion was triggered by the TTTAAA motif, LTR and LINE and healed by a Non Homologous End Joining (NHEJ) mechanism. The genes ATXN7L1 and PDE1C are disrupted by the inversion. PDE1C is responsible for the hydrolysis of the second messenger molecules cAMP and cGMP and is highly expressed in the human heart and certain brain regions. In mice, Pde1c is expressed in migrating neuronal cells within the central nervous system during early embryo development. Although neuronal migration disorder was not seen in our patient, this is the first patient described with haploinsufficiency of PDE1C possibly causing developmental delay.

An infant born to a mother with gestational diabetes presenting with 49,XXXXY syndrome and renal agenesis-a case report.

49,XXXXY is a rare sex chromosome polysomy with an incidence of 1 in 85 000 male births. It has a characteristic triad of mental retardation, skeletal malformation and hypogonadism. This is the first case report of a child with 49,XXXXY syndrome and renal agenesis. This child was referred for genetic testing at 14 years of age due to facial dysmorphism and hypergonadotropic hypogonadism. He had coarse facial features, cryptorchidism of the right testis, genu valgus deformities, and patent ductus arteriosus which are known associations of 49,XXXXY syndrome. He also had agenesis of the right kidney, hydronephrosis of the left kidney with hydroureter which is not a known association of 49,XXXXY syndrome. The patient was the offspring of a mother with gestational diabetes. There is a strong correlation between maternal diabetes and congenital anomalies, especially renal and cardiovascular anomalies. Additionally, it has been noted that gestational diabetes increases the incidence of chromosomal aneuploidies. The teratogenic effects of maternal diabetes during embryogenesis may be the causative factor for the final phenotype of 49,XXXXY syndrome and renal agenesis.