Two alternative methods for the retrieval of somatic cell populations from the mouse ovary.

Many modern techniques employed to uncover the molecular fundamentals underlying biological processes require dissociated cells as their starting point/substrate. Investigations into ovarian endocrinology or folliculogenesis, therefore, necessitate robust protocols for dissociating the ovary into its constituent cell populations. While in the mouse, methods to obtain individual, mature follicles are well-established, the separation and isolation of single cells of all types from early mouse follicles, including somatic cells, has been more challenging. Herein we present two methods for the isolation of somatic cells in the ovary. These methods are suitable for a range of applications relating to the study of folliculogenesis and mouse ovarian development. First, an enzymatic dissociation utilising collagenase and a temporary, primary cell culture step using neonatal mouse ovaries which yields large quantities of granulosa cells from primordial, activating, and primary follicles. Second, a rapid papain dissociation resulting in a high viability single cell suspension of ovarian somatic cells in less than an hour, which can be applied from embryonic to adult ovarian samples. Collectively these protocols can be applied to a broad array of investigations with unique advantages and benefits pertaining to both.

Signal transducer and activator of transcription (STAT) 1 and STAT3 are expressed in the human ovary and have Janus kinase 1-independent functions in the COV434 human granulosa cell line.

Ovarian granulosa cells are fundamental for oocyte maintenance and maturation. Recent studies have demonstrated the importance of members of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathway in the granulosa cell population of mouse and horse ovaries, with perturbation of JAK1 signalling in the mouse shown to impair oocyte maintenance and accelerate primordial follicle activation. The presence and role of the JAK/STAT pathway in human granulosa cells has yet to be elucidated. In this study, expression of JAK1, STAT1 and STAT3 was detected in oocytes and granulosa cells of human ovarian sections from fetal (40 weeks gestation) and premenopausal ovaries (34-41 years of age; n=3). To determine the effects of JAK1 signalling in granulosa cells, the human granulosa-like cell line COV434 was used, with JAK1 inhibition using ruxolitinib. Chemical inhibition of JAK1 in COV434 cells with 100nM ruxolitinib for 72h resulted in significant increases in STAT3 mRNA (P=0.034) and p-Y701-STAT1 protein (P=0.0117), demonstrating a role for JAK1 in modulating STAT in granulosa cells. This study implicates a conserved role for JAK/STAT signalling in human ovary development, warranting further investigation of this pathway in human granulosa cell function.

Medium-term outcomes of the Universal-2 total wrist arthroplasty in patients with rheumatoid arthritis.

AIMS: The aims of this study were to evaluate the clinical and radiological outcomes of the Universal-2 total wrist arthroplasty (TWA) in patients with rheumatoid arthritis. PATIENTS AND METHODS: This was a retrospective review of all 95 Universal-2 TWAs which were performed in our institution between 2003 to 2012 in patients with rheumatoid arthritis. A total of six patients were lost to follow-up and two died of unrelated causes. A total of ten patients had bilateral procedures. Accordingly, 75 patients (85 TWAs) were included in the study. There were 59 women and 16 men with a mean age of 59 years (26 to 86). The mean follow-up was 53 months (24 to 120). Clinical assessment involved recording pain on a visual analogue score, range of movement, grip strength, the Quick Disabilities of the Arm, Shoulder and Hand (DASH) and Wrightington wrist scores. Any adverse effects were documented with particular emphasis on residual pain, limitation of movement, infection, dislocation and the need for revision surgery. Radiographic assessment was performed pre-operatively and at three, six and 12 months post-operatively, and annually thereafter. Arthroplasties were assessed for distal row intercarpal fusion and loosening. Radiolucent zones around the components were documented according to a system developed at our institution. RESULTS: The mean worst pain was 8.1 (3 to 10) pre-operatively and 5.4 (0 to 10) at latest follow-up (p < 0.001). Movements were preserved with mean dorsiflexion of 29o (0 o to 70 o) and palmar flexion of 21o (0o to 50o). The mean grip strength was 4.8 kg (1.7 to 11.5) pre-operatively and 10 kg (0 to 28) at final follow-up (p < 0.001). The mean QuickDASH and Wrightington wrist scores improved from 61 (16 to 91) to 46 (0 to 89) and 7.9 (1.8 to 10) to 5.7 (0 to 7.8) (p < 0.001). A total of six patients (7%) had major complications; three required revision arthroplasty and three an arthrodesis. The Kaplan-Meier probability of survival using removal of the components as the endpoint was 91% at 7.8 years (95% confidence interval 84 to 91). CONCLUSION: The Universal-2 TWA is recommended for use in patients with rheumatoid arthritis. Cite this article: Bone Joint J 2016;98-B:1642-7.

MatOrtho proximal interphalangeal joint arthroplasty: minimum 2-year follow-up.

The MatOrtho proximal interphalangeal replacement is a cementless cobalt-chromium metal-on-polyethylene mobile-bearing surface replacement arthroplasty. The aim of this study is to report the outcome and complications of this implant at a minimum of 2 years follow-up from a single institution. A retrospective case review was performed on all MatOrtho proximal interphalangeal joint replacements performed with a minimum of 2 years follow-up. Patient demographics, diagnosis, implant revision and other surgical interventions were recorded. Subjective and objective outcomes were evaluated at latest follow-up, including pain scores, range of motion, function and radiographic assessment. A total of 109 implants were inserted in 56 patients. Nine implants (six patients) were lost to follow-up. Of the remaining 100 implants, 75 had been undertaken in females. The mean age at time of surgery was 64 years and the principal diagnosis was osteoarthritis in 74%. The mean follow-up was 47 months (range 24-77). Within the group there was a statistically significant diminution in pain. There was also an improvement in functional scores post-operatively. Improvement in range of motion was seen in those joints with a pre-operative range of motion greater than 20°. Radiologically there was no evidence of loosening or of implant subsidence at final follow-up. The revision rate was 13%. Nine joints were revised to the NeuFlex (silicone rubber) prosthesis, three were converted to an arthrodesis and one had exchange of the MatOrtho prosthesis. The survival of the MatOrtho proximal interphalangeal joint arthroplasty was 85% at a minimum of 2-years follow-up. Patients can be advised that the procedure achieves good pain relief, improvement in functional scores and may improve range of motion. We would, however, caution against this implant's use in joints that are either stiff or have significant deformity and/or instability pre-operatively.

Guidelines for the use of cell lines in biomedical research.

Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise.

Pituitary phenotypes of mice lacking the notch signalling ligand delta-like 1 homologue.

The Notch signalling pathway ligand delta-like 1 homologue (Dlk1, also named Pref1) is expressed throughout the developing pituitary and becomes restricted to mostly growth hormone (GH) cells within the adult gland. We have investigated the role of Dlk1 in pituitary development and function from late embryogenesis to adulthood using a mouse model completely lacking the expression of Dlk1. We confirm that Dlk1-null mice are shorter and weigh less than wild-type littermates from late gestation, at parturition and in adulthood. A loss of Dlk1 leads to significant reduction in GH content throughout life, whereas other pituitary hormones are reduced to varying degrees depending on sex and age. Both the size of the pituitary and the proportion of hormone-producing cell populations are unchanged, suggesting that there is a reduction in hormone content per cell. In vivo challenge of mutant and wild-type littermates with growth hormone-releasing hormone and growth hormone-releasing hexapeptide shows that reduced GH secretion is unlikely to account for the reduced growth of Dlk1 knockout animals. These data suggest that loss of Dlk1 gives rise to minor pituitary defects manifesting as an age- and sex-dependent reduction in pituitary hormone contents. However, Dlk1 expression in other tissue is most likely responsible for the weight and length differences observed in mutant animals.

Genetic control of testis development.

Sex determination refers to the decision of the bipotential early gonads to develop as either testes or ovaries during embryogenesis. In mammals, a single genetic trigger involved in this pivotal decision has been identified on the Y chromosome: the testis-determining gene SRY/Sry. During embryogenesis, SRY triggers the differentiation of Sertoli cells from the supporting cell precursor lineage which would otherwise give granulosa cells in ovaries. Several testis-specific events occur after SRY expression and the onset of Sertoli cell differentiation, notably Leydig cell differentiation, testis cord formation, and development of testis-specific vasculature. Although a number of genes involved in these events have been identified, how they relate to Sry action is poorly understood. Furthermore, even at the adult stage, some of these genes retain a key role in maintaining the testicular fate because conditional ablation of the genes leads to adult testis dysgenesis or transdifferentiation into an ovary. This sheds light on mammalian sex-reprogramming, despite the prevailing dogma that postnatal sex change does not occur in mammals. In this review, we summarize our current understanding of genetic pathways of testis determination and differentiation in mammals, particularly in the mouse and the human.

The detection of full thickness rotator cuff tears using ultrasound.

We have examined the accuracy of 143 consecutive ultrasound scans of patients who subsequently underwent shoulder arthroscopy for rotator-cuff disease. All the scans and subsequent surgery were performed by an orthopaedic surgeon using a portable ultrasound scanner in a one-stop clinic. There were 78 full thickness tears which we confirmed by surgery or MRI. Three moderate-size tears were assessed as partial-thickness at ultrasound scan (false negative) giving a sensitivity of 96.2%. One partially torn and two intact cuffs were over-diagnosed as small full-thickness tears by ultrasound scan (false positive) giving a specificity of 95.4%. This gave a positive predictive value of 96.2% and a negative predictive value of 95.4%. Estimation of tear size was more accurate for large and massive tears at 96.5% than for moderate (88.8%) and small tears (91.6%). These results are equivalent to those obtained by several studies undertaken by experienced radiologists. We conclude that ultrasound imaging of the shoulder performed by a sufficiently-trained orthopaedic surgeon is a reliable time-saving practice to identify rotator-cuff integrity.

The future for stem cell research.

Stem cells have offered much hope by promising to greatly extend the numbers and range of patients who could benefit from transplants, and to provide cell replacement therapy to treat debilitating diseases such as diabetes, Parkinson's and Huntington's disease. The issue of stem cell research is politically charged, prompting biologists to begin engaging in ethical debates, and generating in the general public an unusually high level of interest in this aspect of biology. But excitement notwithstanding, there is a long way to go in basic research before new therapies will be established, and now the pressure is on for scientists and clinicians to deliver.

Sox8 gene expression identifies immature glial cells in developing cerebellum and cerebellar tumours.

Sox8 is a member of the E subgroup of Sox genes, the other members of which are Sox9 and Sox10, both of which are implicated in specific human disorders. Recently, Sox8 homologues have been cloned in chick, mouse and human and have been shown to be strongly expressed in the embryonic and adult brain. Nevertheless, the cell types that express Sox8 have not been determined. We show here that Sox8 is expressed in immature glia in the developing cerebellum. Sox8 is also expressed in scattered cells in the cerebellar tumour, medulloblastoma. This gene therefore provides an early glial marker that may provide more detailed insight into the cellular makeup and consequent behaviour of medulloblastomas.

In vivo analysis of the regulation of the anti-Müllerian hormone, as a marker of Sertoli cell differentiation during testicular development, reveals a multi-step process.

Anti-Müllerian hormone (AMH) is a member of the TGF-beta family which elicits its main action during male sex differentiation. This hormone is probably the most convenient marker of Sertoli cell differentiation and maturation throughout testicular development. Studying AMH gene regulation may thus be one way of identifying effectors of Sertoli cell differentiation. To this end we first tried to locate and then to characterise DNA elements responsible for in vivo transcriptional control of AMH expression. We obtained transgenic mice expressing a reporter gene (LacZ), under control of various putative AMH regulatory sequences. Analysis of transgenic animals revealed that activation of the AMH gene probably requires a two-step regulatory process. The first step corresponds to the initial activation of the AMH gene occurring at around 12.0 dpc. It requires the presence of regulatory DNA encompassed within a maximum of 370 bp upstream of the translation start site of the gene, delimited by the presence of an upstream housekeeping gene (SAP-62). Following this initial transient phase, a second phase seems to account for the persistence of AMH gene expression until the onset of puberty. As the 370 bp regulatory region is not sufficient on its own to allow the triggering of this second phase, it seems possible that additional control elements are required for normal AMH expression throughout testicular development. The complete array of regulatory elements remains to be located. Mol. Reprod. Dev. 59:256-264, 2001.

Evidence that the testis determination pathway interacts with a non-dosage compensated, X-linked gene.

In a number of mammals, including mouse and man, it has been shown that at equivalent gestational ages, males are developmentally more advanced than females, even before the gonads form. In mice, although some strains of Y chromosome exert a minor accelerating effect in pre-implantation development, it is a post-implantation effect of the difference in X chromosome constitution that is the major cause of the male/female developmental difference. Thus XX females are retarded in their development by about 1.5 h relative to X(M)O females or XY males; however, they are more advanced than X(P)O females by about 4 h. It has been suggested that this early developmental difference between XX and XY embryos may "weight the dice" in favour of ovarian and testicular development, respectively, although expression of Sry will normally overcome any such bias. Here we test this proposal by comparing the relative frequencies of female, hermaphrodite and male development in X(P)O, XX and X(M)O mice that carry an incompletely penetrant Sry transgene. The results show that testicular tissue develops more frequently in XX,Sry transgenics than in either of the two types of XO transgenics. Thus the incidence of testicular development is affected by X dosage rather than by the developmental hierarchy. This implies there is a non-dosage compensated gene (or genes) on the X chromosome, which interacts with the testis-determining pathway. Since the pseudoautosomal region (PAR) is known to escape X-inactivation, penetrance of the Sry transgene was also assessed in X(M)Y(*X) mice that have two doses of the PAR but have a single dose of all genes proximal to the distal X marker Amel. These mice showed similar levels of testicular development to X(M)O mice with the transgene; thus the non-dosage compensated X gene maps outside the PAR.

Micro-capillary tube in situ hybridisation: a novel method for processing small individual samples.

We have developed a strategy to individually analyse large numbers of small tissue samples by RNA in situ hybridisation. Samples of approximately 0.4 mm x 0.5 mm are processed in rectangular capillary tubes fitted with nylon mesh and glass beads using standard protocols. Eighteen samples can be assayed simultaneously without loss, and background is low. Specifically, mouse Sox2 RNA expression is examined in the chorion of extraembryonic tissue of 7.5 days post-coitum embryos. This technique works equally well for double RNA labelling and could potentially be used for antibody staining of proteins.

Sox2 regulatory sequences direct expression of a (beta)-geo transgene to telencephalic neural stem cells and precursors of the mouse embryo, revealing regionalization of gene expression in CNS stem cells.

Sox2 is one of the earliest known transcription factors expressed in the developing neural tube. Although it is expressed throughout the early neuroepithelium, we show that its later expression must depend on the activity of more than one regionally restricted enhancer element. Thus, by using transgenic assays and by homologous recombination-mediated deletion, we identify a region upstream of Sox2 (-5.7 to -3.3 kb) which can not only drive expression of a (beta)-geo transgene to the developing dorsal telencephalon, but which is required to do so in the context of the endogenous gene. The critical enhancer can be further delimited to an 800 bp fragment of DNA surrounding a nuclease hypersensitive site within this region, as this is sufficient to confer telencephalic expression to a 3.3 kb fragment including the Sox2 promoter, which is otherwise inactive in the CNS. Expression of the 5.7 kb Sox2(beta)-geo transgene localizes to the neural plate and later to the telencephalic ventricular zone. We show, by in vitro clonogenic assays, that transgene-expressing (and thus G418-resistant) ventricular zone cells include cells displaying functional properties of stem cells, i.e. self-renewal and multipotentiality. We further show that the majority of telencephalic stem cells express the transgene, and this expression is largely maintained over two months in culture (more than 40 cell divisions) in the absence of G418 selective pressure. In contrast, stem cells grown in parallel from the spinal cord never express the transgene, and die in G418. Expression of endogenous telencephalic genes was similarly observed in long-term cultures derived from the dorsal telencephalon, but not in spinal cord-derived cultures. Thus, neural stem cells of the midgestation embryo are endowed with region-specific gene expression (at least with respect to some networks of transcription factors, such as that driving telencephalic expression of the Sox2 transgene), which can be inherited through multiple divisions outside the embryonic environment.

Crossover breakpoint mapping identifies a subtelomeric hotspot for male meiotic recombination.

Segregation analysis of CEPH and other pedigrees yielded six paternal crossover breakpoints in the approximately 85 kb interval between the minisatellite loci D16S309 (MS205) and D16S83 (EKMDA2) in 16p13.3. Three crossovers were mapped to within the same small (<3 kb) interval, which does not co-localize with any tandem repeat array or expressed sequence. Haplotyping of loci harbouring single nucleotide polymorphism (SNP) markers in this interval confirmed the exchange of flanking markers in the three recombinant individuals. Sequence analysis revealed the presence of recombination-associated motifs and binding sites for the protein translin. Haplotyping of 108 individuals from three European populations at four loci harbouring SNPs showed substantial linkage equilibrium across this interval. Hence molecular and population genetic data are consistent with the presence of an intense male-specific recombination hotspot at this locus.

Targeted mutagenesis of the endogenous mouse Mis gene promoter: in vivo definition of genetic pathways of vertebrate sexual development.

Mutations were introduced into conserved steroidogenic factor 1 (SF1)- and SOX9-binding sites within the endogenous mouse Mullerian inhibiting substance (Mis) promoter. Male mice homozygous for the mutant SF1-binding site correctly initiated Mis transcription in fetal testes, although at significantly reduced levels. Surprisingly, sufficient MIS was produced to eliminate the MUllerian ducts. In contrast, males homozygous for the mutant SOX9-binding site did not initiate Mis transcription, resulting in pseudohermaphrodites. These studies suggest an essential role for SOX9 in the initiation of Mis transcription, whereas SF1 appears to act as a quantitative regulator of Mis transcript levels, perhaps for influencing non-Mullerian duct tissues. Comparative studies of Mis expression in vertebrates indicate that the Mis promoter receives transcriptional inputs that vary between species but result in the same functional readout.

Sry-negative XX sex reversal in purebred dogs.

The gene responsible for testis induction in normal male mammals is the Y-linked Sry. However, there is increasing evidence that other genes may have testis-determining properties. In XX sex reversal (XXSR), testis tissue develops in the absence of the Y chromosome. Previous polymerase chain reaction (PCR) assays indicated that autosomal recessive XXSR in the American cocker spaniel is Sry-negative. In this study, genomic DNA from the breeding colony of American cocker spaniels and from privately owned purebred dogs were tested by PCR using canine primers for the Sry HMG box and by Southern blots probed with the complete canine Sry coding sequence. Sry was not detected by either method in genomic DNA of affected American cocker spaniels or in the majority (20/21) of affected privately owned purebred dogs. These results confirm that the autosomal recessive form of XXSR in the American cocker spaniel is Sry-negative. In combination with previous studies, this indicates that Sry-negative XXSR occurs in at least 15 dog breeds. The canine disorder may be genetically heterogeneous, potentially with a different mutation in each breed, and may provide several models for human Sry-negative XXSR. A comparative approach to sex determination should be informative in defining the genetic and cellular mechanisms that are common to all mammals.