Transcriptomic profiling of neonatal mouse granulosa cells reveals new insights into primordial follicle activation

The dormant population of ovarian primordial follicles is determined at birth and serves as the reservoir for future female fertility. Yet our understanding of the molecular, biochemical, and cellular processes underpinning primordial follicle activation remains limited. The survival of primordial follicles relies on the correct complement and morphology of granulosa cells, which provide signaling factors essential for oocyte and follicular survival. To investigate the contribution of granulosa cells in the primordial-to-primary follicle transition, gene expression profiles of granulosa cells undergoing early differentiation were assessed in a murine model. Ovaries from C57Bl/6 mice were enzymatically dissociated at time-points spanning the initial wave of primordial follicle activation. Post-natal day (PND) 1 ovaries yielded primordial granulosa cells, and PND4 ovaries yielded a mixed population of primordial and primary granulosa cells. The comparative transcriptome of granulosa cells at these time-points was generated via Illumina NextSeq 500 system, which identified 131 significantly differentially expressed transcripts. The differential expression of eight of the transcripts was confirmed by RT-qPCR. Following biological network mapping via Ingenuity Pathway Analysis, the functional expression of the protein products of three of the differentially expressed genes, namely FRZB, POD1, and ZFX, was investigated with in-situ immunolocalization in PND4 mouse ovaries was investigated. Finally, evidence was provided that Wnt pathway antagonist, secreted frizzled-related protein 3 (FRZB), interacts with a suppressor of primordial follicle activation WNT3A and may be involved in promoting primordial follicle activation. This study highlights the dynamic changes in gene expression of granulosa cells during primordial follicle activation and provides evidence for a renewed focus into the Wnt signaling pathway's role in primordial follicle activation

The association between reproductive health smartphone applications and fertility knowledge of Australian women

Background Previous studies have identified that women living in developed countries have insufficient knowledge of factors which may be contributing to the increasingly high global infertility rates such as maternal age and assisted reproductive technologies. There is a large market of reproductive health smartphone applications, yet little is known about the advantages these apps may confer to users in regards to reproductive health knowledge. Methods An anonymous, online survey of women living in Australia aged 18 and above was open March–June 2018, until ≥200 responses were acquired for statistical power. Respondents answered questions regarding knowledge about general fertility and related factors (age, cyclic fertility, smoking, obesity, miscarriage rate, and success of assisted reproductive technologies). Fertility knowledge was compared in respondents who did or did not use apps relating to female reproductive health. Additionally the functions preferred in reproductive health apps was described by app using respondents. Sociodemographic information was also collected, and relevant data within the dataset was subject to multivariable modelling for the outcome of the fertility knowledge questions. Results Of the 673 respondents that completed the survey, 43.09% reported using mobile phone applications relating to female reproductive health. On average, respondents answered only three of the six fertility knowledge questions correctly. App using respondents were more likely to score better on one question, related to fertility during the menstrual cycle (p < 0.001). App users most commonly reported using the menstrual tracking function in apps (82.4%), which may account for the increased knowledge of cyclic fertility. Conclusions This data provides preliminary evidence toward the usefulness of smartphone applications as a medium for providing information about fertility to women. A limited understanding of one’s own fertility was demonstrated despite being essential for the decision-making of women throughout their reproductive years

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

RBM5 Is a Male Germ Cell Splicing Factor and Is Required for Spermatid Differentiation and Male Fertility

Alternative splicing of precursor messenger RNA (pre-mRNA) is common in mammalian cells and enables the production of multiple gene products from a single gene, thus increasing transcriptome and proteome diversity. Disturbance of splicing regulation is associated with many human diseases; however, key splicing factors that control tissue-specific alternative splicing remain largely undefined. In an unbiased genetic screen for essential male fertility genes in the mouse, we identified the RNA binding protein RBM5 (RNA binding motif 5) as an essential regulator of haploid male germ cell pre-mRNA splicing and fertility. Mice carrying a missense mutation (R263P) in the second RNA recognition motif (RRM) of RBM5 exhibited spermatid differentiation arrest, germ cell sloughing and apoptosis, which ultimately led to azoospermia (no sperm in the ejaculate) and male sterility. Molecular modelling suggested that the R263P mutation resulted in compromised mRNA binding. Within the adult mouse testis, RBM5 localises to somatic and germ cells including spermatogonia, spermatocytes and round spermatids. Through the use of RNA pull down coupled with microarrays, we identified 11 round spermatid-expressed mRNAs as putative RBM5 targets. Importantly, the R263P mutation affected pre-mRNA splicing and resulted in a shift in the isoform ratios, or the production of novel spliced transcripts, of most targets. Microarray analysis of isolated round spermatids suggests that altered splicing of RBM5 target pre-mRNAs affected expression of genes in several pathways, including those implicated in germ cell adhesion, spermatid head shaping, and acrosome and tail formation. In summary, our findings reveal a critical role for RBM5 as a pre-mRNA splicing regulator in round spermatids and male fertility. Our findings also suggest that the second RRM of RBM5 is pivotal for appropriate pre-mRNA splicing.This work was supported by grants from the National Health and Medical Research Council (NHMRC) to DJ (#606503); the Australian Research Council (ARC) to MKO and CJO; the New South Wales Cancer Council, Cancer Institute New South Wales, Banque Nationale de Paris-Paribas Australia and New Zealand, RT Hall Trust, and the National Breast Cancer Foundation to CJO. DJ was an NHMRC Peter Doherty Postdoctoral Fellow (#384297). MKO and CJO are NHMRC Senior Research Fellows (#545805, #481310). CCG is an NHMRC Australia Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Chronic testicular Chlamydia muridarum infection impairs mouse fertility and offspring development

With approximately 131 million new genital tract infections occurring each year, Chlamydia is the most common sexually transmitted bacterial pathogen worldwide. Male and female infections occur at similar rates and both cause serious pathological sequelae. Despite this, the impact of chlamydial infection on male fertility has long been debated, and the effects of paternal chlamydial infection on offspring development are unknown. Using a male mouse chronic infection model, we show that chlamydial infection persists in the testes, adversely affecting the testicular environment. Infection increased leukocyte infiltration, disrupted the blood:testis barrier and reduced spermiogenic cell numbers and seminiferous tubule volume. Sperm from infected mice had decreased motility, increased abnormal morphology, decreased zona-binding capacity, and increased DNA damage. Serum anti-sperm antibodies were also increased. When both acutely and chronically infected male mice were bred with healthy female mice, 16.7% of pups displayed developmental abnormalities. Female offspring of chronically infected sires had smaller reproductive tracts than offspring of noninfected sires. The male pups of infected sires displayed delayed testicular development, with abnormalities in sperm vitality, motility, and sperm-oocyte binding evident at sexual maturity. These data suggest that chronic testicular Chlamydia infection can contribute to male infertility, which may have an intergenerational impact on sperm quality

Gemcitabine Plus Bevacizumab Compared With Gemcitabine Plus Placebo in Patients With Advanced Pancreatic Cancer: Phase III Trial of the Cancer and Leukemia Group B (CALGB 80303)

The combination of gemcitabine plus bevacizumab produced a 21% response rate and a median survival of 8.8 months in a multicenter phase II trial in patients with metastatic pancreatic cancer. These encouraging data led Cancer and Leukemia Group B (CALGB) to conduct a double-blind, placebo-controlled, randomized phase III trial of gemcitabine/bevacizumab versus gemcitabine/placebo in advanced pancreatic cancer patients

Common errors of reasoning in child protection work

Objective: repeated public inquiries into child abuse tragedies in Britain demonstrate the level of public concern about the services designed to protect children. These inquiries identify faults in professionals’ practice but the similarities in their findings indicate that they are having insufficient impact on improving practice. This study is based on the hypothesis that the recurrent errors may be explicable as examples of the typical errors of human reasoning identified by psychological research. Methods: the sample comprised all child abuse inquiry reports published in Britain between 1973 and 1994 (forty-five in total). Using a content analysis and a framework derived from psychological research on reasoning, a study was made of the reasoning of the professionals involved and the findings of the inquiries. Results: it was found that professionals based assessments of risk on a narrow range of evidence. It was biased towards the information readily available to them, overlooking significant data known to other professionals. The range was also biased towards the more memorable data, that is, towards evidence that was vivid, concrete, arousing emotion and either the first or last information received. The evidence was also often faulty, due, in the main, to biased or dishonest reporting or errors in communication. A critical attitude to evidence was found to correlate with whether or not the new information supported the existing view of the family. A major problem was that professionals were slow to revise their judgements despite a mounting body of evidence against them. Conclusions: errors in professional reasoning in child protection work are not random but predictable on the basis of research on how people intuitively simplify reasoning processes in making complex judgements. These errors can be reduced if people are aware of them and strive consciously to avoid them. Aids to reasoning need to be developed that recognise the central role of intuitive reasoning but offer methods for checking intuitive judgements more rigorously and systematically

Ovarian ageing : where are we now? and where to next?

From the mid-1980s, the scientific community has demonstrated irrefutable evidence for the age-related decline in female fertility (Menken et al., 1986) [1], supporting observations in animals and humans of maternal ageing, increased incidence of miscarriage and congenital abnormality dating back to the mid-20th Century (Warburton, 1970) [2]. Although, it was not until 2005 that a report arising from the European Society of Human Reproduction and Embryology sought to address ovarian ageing as the primary contributing factor to this decline and its profound influence on the success of assisted reproduction. Over the resulting two decades, researchers have endeavoured to define and quantify the genetic, molecular and cellular factors directly responsible for ovarian ageing and subsequently female age-related infertility, seeking to ultimately improve diagnostics and treatments. In this review, we undertake a closer investigation of the ‘usual suspects’ and speculate on the role of synergistic inflammaging and finally address the question of whether in 2020 we are really any closer to finding the culprits underpinning the ageing ovary

Regulation of Bile Salt Transport in Rat Liver: EVIDENCE THAT INCREASED MAXIMUM BILE SALT SECRETORY CAPACITY IS DUE TO INCREASED CHOLIC ACID RECEPTORS

Expansion of the bile salt pool size in rats increases maximum excretory capacity for taurocholate. We examined whether increased bile salt transport is due to recruitment of centrolobular transport units or rather to adaptive changes in the hepatocyte. Daily sodium cholate (100 mg/100 g body wt) was administered orally to rats. This treatment was well tolerated for at least 4 d and produced an 8.2-fold expansion of the bile salt pool. This expanded pool consisted predominently (99%) of cholic and deoxycholic acids. Significantly increased bile salt transport was not observed until 16 h after bile acid loading, and maximum elevations of transport capacity to 2.3-fold of control required ∼2 d. In contrast, maximum sulfobromophthalein excretion rates increased 2.2-fold as early as 4 h and actually fell to 1.5-fold increase at 4 d. We studied the possibility that this adaptive increase in bile salt secretory transport was due to changes in canalicular surface membrane area, lipid composition, or increased number of putative carriers. Canalicular membrane protein recovery and the specific activities of leucine aminopeptidase, Mg(++)-ATPase and 5′-nucleotidase activities were unaltered by bile salt pool expansion. The content of free and esterified cholesterol and total phospholipids was unchanged in liver surface membrane fractions compared with control values. In contrast, sodium cholate administration selectively increased specific [(14)C]cholic acid binding sites twofold in liver surface membrane fractions. Increased numbers of [(14)C]cholic acid receptors (a) was associated with the time-dependent increase in bile salt transport, and (b) was selective for the taurine conjugate of cholate and (c) was reduced by chenodeoxycholate. Changes in bile acid binding sites 16 h following taurocholate and chenodeoxycholate and the lack of change with glycocholate was associated with comparable changes in bile salt transport. In conclusion, selective bile salts increase bile salt transport in the liver through an adaptive increase in the density of putative bile acid carriers in liver surface membrane