In biomedicine and many other fields, there are growing concerns around the reproducibility of research findings, with many researchers being unable to replicate their own or others’ results. This raises important questions as to the validity and usefulness of much published research. In this review, we aim to engage researchers in the issue of research reproducibility and equip them with the necessary tools to increase the reproducibility of their research. We first highlight the causes and potential impact of non-reproducible research and emphasise the benefits of working reproducibly for the researcher and broader research community. We address specific targets for improvement and steps that individual researchers can take to increase the reproducibility of their work. We next provide recommendations for improving the design and conduct of experiments, focusing on in vivo animal experiments. We describe common sources of poor internal validity of experiments and offer practical guidance for limiting these potential sources of bias at different experimental stages, as well as discussing other important considerations during experimental design. We provide a list of key resources available to researchers to improve experimental design, conduct, and reporting. We then discuss the importance of open research practices such as study preregistration and the use of preprints and describe recommendations around data management and sharing. Our review emphasises the importance of reproducible work and aims to empower every individual researcher to contribute to the reproducibility of research in their field.

Neuroactive steroids can rapidly regulate multiple physiological functions in the central and peripheral nervous systems. The aims of the present study were to determine whether allopregnanolone (ALLO), administered in low nanomolar and high micromolar concentrations, can: (i) induce changes in the ovarian progesterone (P₄) and estradiol (E₂) release; (ii) modify the ovarian mRNA expression of Hsd3b1 (3β-hydroxysteroid dehydrogenase, 3β-HSD)3β-, Akr1c3 (20α-hydroxysteroid dehydrogenase, 20α-HSD), and Akr1c14 (3α-hydroxy steroid oxidoreductase, 3α-HSOR)); and (iii) modulate the ovarian expression of progesterone receptors A and B, α and β estrogenic receptors, luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR). To further characterize ALLO peripheral actions, the effects were evaluated using a superior mesenteric ganglion–ovarian nervous plexus–ovary (SMG–ONP–O) and a denervated ovary (DO) systems. ALLO SMG administration increased P₄ concentration in the incubation liquid by decreasing ovarian 20α-HSD mRNA, and it also increased ovarian 3α-HSOR mRNA expression. In addition, ALLO neural peripheral modulation induced an increase in the expression of ovarian LHR, PRA, PRB, and ERα. Direct ALLO administration to the DO decreased E₂ and increased P₄ concentration in the incubation liquid. The mRNA expression of 3β-HSD decreased and 20α-HSD increased. Further, ALLO in the OD significantly changed ovarian FSHR and PRA expression. This is the first evidence of ALLO’s direct effect on ovarian steroidogenesis. Our results provide important insights about how this neuroactive steroid interacts both with the PNS and the ovary, and these findings might help devise some of the pleiotropic effects of neuroactive steroids on female reproduction. Moreover, ALLO modulation of ovarian physiology might help uncover novel treatment approaches for reproductive diseases.

In commemoration of 100 years since the discovery of glucagon, we review current knowledge about the human α cell. Alpha cells make up 30–40% of human islet endocrine cells and play a major role in regulating whole-body glucose homeostasis, largely through the direct actions of their main secretory product – glucagon – on peripheral organs. Additionally, glucagon and other secretory products of α cells, namely acetylcholine, glutamate, and glucagon-like peptide-1, have been shown to play an indirect role in the modulation of glucose homeostasis through autocrine and paracrine interactions within the islet. Studies of glucagon’s role as a counterregulatory hormone have revealed additional important functions of the α cell, including the regulation of multiple aspects of energy metabolism outside that of glucose. At the molecular level, human α cells are defined by the expression of conserved islet-enriched transcription factors and various enriched signature genes, many of which have currently unknown cellular functions. Despite these common threads, notable heterogeneity exists amongst human α cell gene expression and function. Even greater differences are noted at the inter-species level, underscoring the importance of further study of α cell physiology in the human context. Finally, studies on α cell morphology and function in type 1 and type 2 diabetes, as well as other forms of metabolic stress, reveal a key contribution of α cell dysfunction to dysregulated glucose homeostasis in disease pathogenesis, making targeting the α cell an important focus for improving treatment.

Leukaemia inhibitory factor (LIF) is a cytokine belonging to the interleukin-6 family that is important at the reproductive level in the uterine implantation process. However, there is very little evidence regarding its effect at the ovarian level. The aim of this work was to study the local involvement of the LIF/LIFRβ system in follicular development and steroidogenesis in rat ovaries. To carry out this research, LIF/LIFR/GP130 transcript and protein levels were measured in fertile and sub-fertile rat ovaries, and in vitro experiments were performed to assess STAT3 activation. Then, in in vivo experiments, LIF was administered chronically and locally for 28 days to the ovaries of rats by means of an osmotic minipump to enable us to evaluate the effect on folliculogenesis and steroidogenesis. It was determined by quantitative polymerase chain reaction and western blot that LIF and its receptors are present in fertile and sub-fertile ovaries and that LIF varies during the oestrous cycle, being higher during the oestrus and meta/dioestrus stages. In addition to this, it was found that LIF can activate STAT3 pathways and cause pSTAT3 formation. It was also observed that LIF decreases the number and size of preantral and antral follicles without altering the number of atretic antral follicles and can increase the number of corpora lutea, with a notable increase in the levels of progesterone (P4). It is therefore possible to infer that LIF exerts an important effect in vivo on folliculogenesis, ovulation and steroidogenesis, specifically the synthesis of P4.

Obesity is caused by imbalanced energy intake and expenditure. Excessive energy intake and storage in adipose tissues are associated with many diseases. Several studies have demonstrated that vascular growth endothelial factor B (VEGFB) deficiency induces obese phenotypes. However, the roles of VEGFB isoforms VEGFB₁₆₇ and VEGFB₁₈₆ in adipose tissue development and function are still not clear. In this study, genetic mouse models of adipose-specific VEGFB₁₆₇ and VEGFB₁₈₆ overexpression (aP2-Vegfb₁₆₇ ^tg/+ and aP2-Vegfb₁₈₆ ^tg/+ ) were generated and their biologic roles were investigated. On regular chow, adipose-specific VEGFB₁₈₆ is negatively associated with white adipose tissues (WATs) and positively regulates brown adipose tissues (BATs). VEGFB₁₈₆ upregulates energy metabolism and metabolism-associated genes. In contrast, VEGFB₁₆₇ has a nominal role in adipose development and function. On high-fat diet, VEGFB₁₈₆ expression can reverse the phenotypes of VEGFB deletion. VEGFB₁₈₆ overexpression upregulates BAT-associated genes and downregulates WAT-associated genes. VEGFB₁₈₆ and VEGFB₁₆₇ have very distinct roles in the regulation of adipose development and energy metabolism. As a key regulator of adipose tissue development and energy metabolism, VEGFB₁₈₆ may be a target for obesity prevention and treatment.

Modeling preeclampsia remains difficult due to the nature of the disease and the unique characteristics of the human placenta. Members of the Hominidae superfamily have a villous hemochorial placenta that is different in structure from those of other therian mammals, including the mouse hemochorial placenta, making this common animal model less ideal for studying this disease. Human placental tissues delivered from pregnancies complicated by preeclampsia are excellent for assessing the damage the disease causes but cannot answer how or when the disease begins. Symptoms of preeclampsia manifest halfway through pregnancy or later, making it currently impossible to identify preeclampsia in human tissues obtained from an early stage of pregnancy. Many animal and cell culture models recapitulate various aspects of preeclampsia, though none can on its own completely capture the complexity of human preeclampsia. It is particularly difficult to uncover the cause of the disease using models in which the disease is induced in the lab. However, the many ways by which preeclampsia-like features can be induced in a variety of laboratory animals are consistent with the idea that preeclampsia is a two-stage disease, in which a variety of initial insults may lead to placental ischemia, and ultimately systemic symptoms. The recent development of stem cell-based models, organoids, and various coculture systems have brought in vitro systems with human cells ever closer to recapitulating in vivo events that lead to placental ischemia.

Based on biological sex, the consequential health outcomes from exposures to environmental chemicals or toxicants can differ in disease pathophysiology, progression, and severity. Due to basal differences in cellular and molecular processes resulting from sexual dimorphism of organs including the liver and additional factors influencing ‘gene–environment’ interactions, males and females can exhibit different responses to toxicant exposures. Associations between environmental/occupational chemical exposures and fatty liver disease (FLD) have been well-acknowledged in human epidemiologic studies and their causal relationships demonstrated in experimental models. However, studies related to sex differences in liver toxicology are still limited to draw any inferences on sex-dependent chemical toxicity. The purpose of this review is to highlight the present state of knowledge on the existence of sex differences in toxicant-associated FLD (TAFLD), discuss potential underlying mechanisms driving these differences, implications of said differences on disease susceptibility, and emerging concepts. Chemicals of interest include various categories of pollutants that have been investigated in TAFLD, namely persistent organic pollutants, volatile organic compounds, and metals. Insight into research areas requiring further development is also discussed, with the objective of narrowing the knowledge gap on sex differences in environmental liver diseases. Major conclusions from this review exercise are that biological sex influences TAFLD risks, in part due to (i) toxicant disruption of growth hormone and estrogen receptor signaling, (ii) basal sex differences in energy mobilization and storage, and (iii) differences in chemical metabolism and subsequent body burden. Finally, further sex-dependent toxicological assessments are warranted for the development of sex-specific intervention strategies.

Although originally characterised as proteins involved in the control of reproductive function, activins, and to a lesser degree inhibins, are also important regulators of homeostasis in extragonadal tissues. Accordingly, disrupted inhibin/activin expression can have detrimental effects not only on fertility and fecundity but also on the regulation of muscle, fat and bone mass. Indeed, only recently, two complementary mouse models of inhibin designed to lack bioactivity/responsiveness revealed that inhibin A/B deficiency during pregnancy restricts embryo and fetal survival. Conversely, hyper-elevated levels of activin A/B, as are frequently observed in patients with advanced cancers, can not only promote gonadal tumour growth but also cancer cachexia. As such, it is not surprising that inhibin/activin genetic variations or altered circulating levels have been linked to reproductive disorders and cancer. Whilst some of the detrimental health effects associated with disrupted inhibin/activin levels can be attributed to accompanied changes in circulating follicle-stimulating hormone (FSH) levels, there is now abundant evidence that activins, in particular, have fundamental FSH-independent tissue homeostatic roles. Increased understanding of inhibin/activin activity, garnered over several decades, has enabled the development of targeted therapies with applications for both reproductive and extra-gonadal tissues. Inhibin- or activin-targeted technologies have been shown not just to enhance fertility and fecundity but also to reduce disease severity in models of cancer cachexia. Excitingly, these technologies are likely to benefit human medicine and be highly valuable to animal breeding and veterinary programmes.

Patients suffering from chronic kidney disease (CKD) often experience bone loss and arterial calcifications. It is unclear if hypogonadism contributes to the development of these complications and whether androgen therapy might prevent them. Male adult rats were randomized into four groups. The first group received standard chow (control), while three other groups were fed a 0.25% adenine/low vitamin K diet (CKD). Two CKD groups were treated with testosterone or dihydrotestosterone (DHT), whereas the control group and one CKD group received vehicle (VEH). CKD animals had 10-fold higher serum creatinine and more than 15-fold higher parathyroid hormone levels compared to controls. Serum testosterone levels were more than two-fold lower in the CKDVEH group compared to control + VEH and CKD + testosterone groups. Seminal vesicle weight was reduced by 50% in CKDVEH animals and restored by testosterone and DHT. CKD animals showed a low bone mass phenotype with decreased trabecular bone volume fraction and increased cortical porosity, which was not rescued by androgen treatment. Aortic calcification was much more prominent in CKD animals and not unequivocally prevented by androgens. Messenger RNA expression of the androgen receptor-responsive genes Acta1 and Col1a1 was reduced by CKD and stimulated by androgen treatment in levator ani muscle but not in the bone or aortic tissue. We conclude that adenine-induced CKD results in the development of hypogonadism in male rats. Androgen therapy is effective in restoring serum testosterone levels and androgen-sensitive organ weights but does not prevent bone loss or arterial calcifications, at least not in the presence of severe hyperparathyroidism.

Diabetes-related foot disease (DFD), defined as ulceration, infection or destruction of tissues of the foot in a person with current or previously diagnosed diabetes mellitus, is associated with a heavy burden for both patients and the healthcare system with high morbidity, mortality and costs. Improved outcomes for people with DFD are achieved with an interdisciplinary approach and adherence to best practice clinical guidelines; however, in the Australian context, the vastness of the country presents unique challenges in achieving optimal outcomes for all people with DFD, with variation in service delivery, availability and accessibility between metropolitan, rural and remote areas. Aboriginal and Torres Strait Islander Australians and people with diabetes living in rural and remote areas experience higher rates of lower-extremity amputation, and further efforts and resources are required to improve outcomes for these high-risk groups. In recent years, there have been advances in knowledge, including the understanding of the pathogenesis of diabetes-related peripheral neuropathy, genetic polymorphisms and mechanisms of disease associated with acute Charcot neuroarthropathy, biomarkers and potential mediators of diabetes-related foot ulcer (DFU) healing, the microbiology and microbiome profile of DFUs, pressure assessment and management as well as an expanded understanding of DFU sequelae and comorbidities. In this review, we describe new insights into pathophysiology, sequelae and comorbidities of DFD with a focus on basic and translational aspects and contributions to the field from Australian and New Zealand DFD researchers.