The Role of PLAG1 in Mouse Brain Development and Neurogenesis.

The pleomorphic adenoma gene 1 (Plag1) is a transcription factor involved in the regulation of growth and cellular proliferation. Here, we report the spatial distribution and functional implications of PLAG1 expression in the adult mouse brain. We identified Plag1 promoter-dependent ß-galactosidase expression in various brain structures, including the hippocampus, cortex, choroid plexus, subcommisural organ, ependymal cells lining the third ventricle, medial and lateral habenulae and amygdala. We noted striking spatial-restriction of PLAG1 within the cornu ammonis (CA1) region of the hippocampus and layer-specific cortical expression, with abundant expression noted in all layers except layer 5. Furthermore, our study delved into the role of PLAG1 in neurodevelopment, focusing on its impact on neural stem/progenitor cell proliferation. Loss of Plag1 resulted in reduced proliferation and decreased production of neocortical progenitors in vivo, although ex vivo neurosphere experiments revealed no cell-intrinsic defects in the proliferative or neurogenic capacity of Plag1-deficient neural progenitors. Lastly, we explored potential target genes of PLAG1 in the cortex, identifying that Neurogenin 2 (Ngn2) was significantly downregulated in Plag1-deficient mice. In summary, our study provides novel insights into the spatial distribution of PLAG1 expression in the adult mouse brain and its potential role in neurodevelopment. These findings expand our understanding of the functional significance of PLAG1 within the brain, with potential implications for neurodevelopmental disorders and therapeutic interventions.

Crucial Convolution: Genetic and Molecular Mechanisms of Coiling during Epididymis Formation and Development in Embryogenesis.

As embryonic development proceeds, numerous organs need to coil, bend or fold in order to establish their final shape. Generally, this occurs so as to maximise the surface area for absorption or secretory functions (e.g., in the small and large intestines, kidney or epididymis); however, mechanisms of bending and shaping also occur in other structures, notably the midbrain-hindbrain boundary in some teleost fish models such as zebrafish. In this review, we will examine known genetic and molecular factors that operate to pattern complex, coiled structures, with a primary focus on the epididymis as an excellent model organ to examine coiling. We will also discuss genetic mechanisms involving coiling in the seminiferous tubules and intestine to establish the final form and function of these coiled structures in the mature organism.

Effect of Pleomorphic Adenoma Gene 1 Deficiency on Selected Behaviours in Adult Mice.

The proto-oncogene pleomorphic adenoma gene 1 (Plag1) encodes a zinc finger transcription factor. PLAG1 is part of the high motility group AT hook-2 (HGMA2)-PLAG1-insulin-like growth factor 2 (IGF2) pathway that, when disrupted, leads to Silver-Russell syndrome, a severe form of intrauterine growth restriction. With little known about PLAG1's role in normal physiology, this study is the first to characterise the behavioural phenotype of PLAG1-deficient mice. Mice were tested for differences in circadian locomotor activity and body temperature, sleep-like behaviour, anxiety-like behaviour, cognition, social behaviour, and sensorimotor gating. Overall, the behavioural phenotype of the Plag1 knock-out (KO) mice was mild: no significant differences were seen in circadian activity levels, locomotion, object recognition, spatial memory or sociability compared to wild-type mice. However, the cued test of fear conditioning, prepulse inhibition of the startle response and Preyer's reflex test suggest that Plag1 KO mice may have a hearing impairment. This implies that PLAG1 plays an important role in proper functioning and/or development of the neural circuitry behind the auditory processes or interacts with genes involved in those processes.

A pioneer calf foetus microbiome.

Foetus sterility until parturition is under debate due to reports of microorganisms in the foetal environment and meconium. Sufficient controls to overcome sample contamination and provide direct evidence of microorganism viability in the pre-rectal gastrointestinal tract (GIT) have been lacking. We conducted molecular and culture-based analyses to investigate the presence of a microbiome in the foetal GIT of calves at 5, 6 and 7 months gestation, while controlling for contamination. The 5 components of the GIT (ruminal fluid, ruminal tissue, caecal fluid, caecal tissue and meconium) and amniotic fluid were found to contain a pioneer microbiome of distinct bacterial and archaeal communities. Bacterial and archaeal richness varied between GIT components. The dominant bacterial phyla in amniotic fluid differed to those in ruminal and caecal fluids and meconium. The lowest bacterial and archaeal abundances were associated with ruminal tissues. Viable bacteria unique to the ruminal fluids, which were not found in the controls from 5, 6 and 7 months gestation, were cultured, subcultured, sequenced and identified. We report that the foetal GIT is not sterile but is spatially colonised before birth by a pioneer microbiome.

Transcriptome analysis of the epididymis from Plag1 deficient mice suggests dysregulation of sperm maturation and extracellular matrix genes.

BACKGROUND: The transcription factor pleomorphic adenoma gene 1 (PLAG1) is required for male fertility. Mice deficient in PLAG1 exhibit decreased sperm motility and abnormal epididymal tubule elongation and coiling, indicating impaired sperm maturation during epididymal transit. However, the downstream transcriptomic profile of the Plag1 knockout (KO; Plag1-/- ) murine epididymis is currently unknown. RESULTS: In this study, the PLAG1-dependent epididymal transcriptome was characterised using RNA sequencing. Several genes important for the control of sperm maturation, motility, capacitation and the acrosome reaction were dysregulated in Plag1-/- mice. Surprisingly, several cell proliferation genes were upregulated, and Ki67 analysis indicated that cell proliferation is aberrantly upregulated in the cauda epididymis stroma of Plag1-/- mice. Gene ontology analysis showed an overall upregulation of genes encoding extracellular matrix components, and an overall downregulation of genes encoding metalloendopeptidases in the epididymides from Plag1-/- mice. CONCLUSION: Together, these results suggest a defect in the epididymal extracellular matrix in Plag1-/- mice. These results imply that in addition to maintaining epididymal integrity directly, PLAG1 may also regulate several genes involved in the regulation of sperm maturation and capacitation. Moreover, PLAG1 may also be involved in regulating tissue homeostasis and ensuring proper structure and maintenance of the extracellular matrix in the epididymis.

Deficiency of the transcription factor PLAG1 results in aberrant coiling and morphology of the epididymis.

Mice deficient in the transcription factor pleomorphic adenoma gene 1 (PLAG1) exhibit reproductive issues that are characterized, in part, by decreased progressive sperm motility in the male. However, the underlying cause of this impairment is unknown. As epididymal transit is critical for sperm maturation and motility, the morphology of the epididymis of Plag1-deficient mice was investigated and the spatial expression patterns of PLAG1 protein and mRNA were identified. Using X-gal staining and in situ hybridization, PLAG1 was shown to be widely expressed in both the epithelium and stroma in all regions of the mouse epididymis. Interestingly, the X-gal staining pattern was markedly different in the cauda, where it could be suggestive of PLAG1 secretion into the epididymal lumen. At all ages investigated, the morphology of epididymides from Plag1 knockout (KO) mice was aberrant; the tubule failed to elongate and coil, particularly in the corpus and cauda, and the cauda was malformed, lacking its usual bulbous shape. Moreover, the epididymides from Plag1 KO mice were significantly reduced in size relative to body weight. In 20% of Plag1-deficient mice, the left testicle and epididymis were lacking. The impaired morphogenesis of the epididymal tubule is likely to be a major contributing factor to the fertility problems observed in male Plag1-deficient mice. These results also establish PLAG1 as an important regulator of male reproduction, not only through its involvement in testicular sperm production, but also via its role in the development and function of the epididymis.

Interrogating the Grainyhead-like 2 (Grhl2) genomic locus identifies an enhancer element that regulates palatogenesis in mouse.

The highly-conserved Grainyhead-like (Grhl) transcription factors are critical regulators of embryogenesis that regulate cellular survival, proliferation, migration and epithelial integrity, especially during the formation of the craniofacial skeleton. Family member Grhl2 is expressed throughout epithelial tissues during development, and loss of Grhl2 function leads to significant defects in neurulation, abdominal wall closure, formation of the face and fusion of the maxilla/palate. Whereas numerous downstream target genes of Grhl2 have been identified, very little is known about how this crucial developmental transcription factor itself is regulated. Here, using in silico and in utero expression analyses and functional deletion in mice, we have identified a novel 2.4 â€‹kb enhancer element (mm1286) that drives reporter gene expression in a pattern that strongly recapitulates endogenous Grhl2 in the craniofacial primordia, modulates Grhl2 expression in these tissues, and augments Grhl2-mediated closure of the secondary palate. Deletion of this genomic element, in the context of inactivation of one allele of Grhl2 (through generation of double heterozygous Grhl2+/-;mm1286+/- mice), results in a significant predisposition to palatal clefting at birth. Moreover, we found that a highly conserved 325 bp region of mm1286 is both necessary and sufficient for mediating the craniofacial-specific enhancer activity of this region, and that an extremely well-conserved 12-bp sequence within this element (CTGTCAAACAGGT) substantially determines full enhancer function. Together, these data provide valuable new insights into the upstream genomic regulatory landscape responsible for transcriptional control of Grhl2 during palatal closure.

Transcriptional regulation of the chicken CRHR2 gene by pituitary transcription factors.

Corticotropin-releasing hormone (CRH) is known to act as a potent thyrotropin-releasing factor in non-mammalian species such as chicken and bullfrog. This interaction is mediated by type 2 CRH receptors (CRHR2) expressed by the thyrotropes in the pituitary gland. However, the response elements (REs) and their corresponding transcription factors (TFs) that control CRHR2 expression in thyrotropes are not known. Since thyrotrope-specific expression of the ß-subunit of thyrotropin is synergistically stimulated by the co-expression of POU1F1 and GATA2, we hypothesised that in non-mammalian vertebrates like chicken, CRHR2 expression is controlled by the same TFs and that their REs are present in the chicken CRHR2 gene promoter. In situ hybridisation and immunohistochemistry suggest that chicken thyrotropes, like those of mammals, express the mRNAs for the TFs GATA2, POU1F1 and PITX1, but not NR5A1. Using luciferase reporter assays, we show that both GATA2 and PITX1 can activate the promoter of CRHR2, but PITX1 requires a functional GATA2 RE to be present. POU1F1 alone did not affect promoter activity, but synergistically increased the effect of GATA2. Promoter deletion analysis and mutagenesis showed that essential GATA2 and PITX1 REs are located between 116 and 198 bp upstream of the start codon. These REs are highly conserved in non-mammalian species. Additionally, NR5A1 (steroidogenic factor 1) suppressed both GATA2- and PITX1-induced promoter activity and may therefore play a role in restricting CRHR2 expression in gonadotropes. We conclude that the expression of CRHR2 in chicken thyrotropes is stimulated by GATA2 with interactions with POU1F1 and PITX1, in the absence of NR5A1.

An idea to explore: Genotyping bull sperm to introduce basic molecular biology techniques in an animal science course.

The laboratory exercise described here aims to provide a relevant context for learning basic DNA techniques in an introductory animal science course at tertiary level. In two 4-hr laboratory sessions, students assess the suitability of bulls for inclusion in a gene-assisted selection program for A2 ß-casein by genotyping commercial bull sperm. Sperm cells are lysed to extract the genomic DNA, and PCR with primers for the ß-casein gene is performed. Using the principle of amplification-created restriction sites, restriction digestion with TaqI can be used to distinguish between the A1 allele and the A2 allele of the gene. Cut PCR amplicons are separated by gel electrophoresis to evaluate the genotype of each bull. Students then write a diagnostic report with accompanying letter to their fictional client, explaining the DNA test, and interpreting the results. © 2019 International Union of Biochemistry and Molecular Biology, 47(6):708-711, 2019.

Pleomorphic Adenoma Gene 1 Is Needed For Timely Zygotic Genome Activation and Early Embryo Development.

Pleomorphic adenoma gene 1 (PLAG1) is a transcription factor involved in cancer and growth. We discovered a de novo DNA motif containing a PLAG1 binding site in the promoters of genes activated during zygotic genome activation (ZGA) in human embryos. This motif was located within an Alu element in a region that was conserved in the murine B1 element. We show that maternally provided Plag1 is needed for timely mouse preimplantation embryo development. Heterozygous mouse embryos lacking maternal Plag1 showed disrupted regulation of 1,089 genes, spent significantly longer time in the 2-cell stage, and started expressing Plag1 ectopically from the paternal allele. The de novo PLAG1 motif was enriched in the promoters of the genes whose activation was delayed in the absence of Plag1. Further, these mouse genes showed a significant overlap with genes upregulated during human ZGA that also contain the motif. By gene ontology, the mouse and human ZGA genes with de novo PLAG1 motifs were involved in ribosome biogenesis and protein synthesis. Collectively, our data suggest that PLAG1 affects embryo development in mice and humans through a conserved DNA motif within Alu/B1 elements located in the promoters of a subset of ZGA genes.

Evolutionary origin of the type 2 corticotropin-releasing hormone receptor γ splice variant.

Many G protein-coupled receptors have splice variants, with potentially different pharmaceutical properties, expression patterns and roles. The human brain expresses three functional splice variants of the type 2 corticotropin-releasing hormone: CRHR2α, -ß and -γ. CRHR2γ has only been reported in humans, but its phylogenetic distribution, and how and when during mammalian evolution it arose, is unknown. Based on genomic sequence analyses, we predict that a functional CRHR2γ is present in all Old World monkeys and apes, and is unique to these species. CRHR2γ arose by exaptation of an intronic sequence-already present in the common ancestor of primates and rodents-after retrotransposition of a short interspersed nuclear element (SINE) and mutations that created a 5' donor splice site and in-frame start codon, 32-43 million years ago. The SINE is not part of the coding sequence, only of the 5' untranslated region and may therefore play a role in translational regulation. Putative regulatory elements and an alternative transcriptional start site were added earlier to this genomic locus by a DNA transposon. The evolutionary history of CRHR2γ confirms some of the earlier reported principles behind the "birth" of alternative exons. The functional significance of CRHR2γ, particularly in the brain, remains to be showed.

Bone marrow fat analysis as a diagnostic tool to document ante-mortem starvation.

Veterinary diagnostic clinicians are increasingly presented with emaciated animals involved in suspected neglect cases. A rise in public awareness and media attention towards animal welfare, combined with changes in legislation and a demand for a higher standard of evidence be presented in animal neglect cases submitted for prosecutions, have created a need for an objective measurement of starvation, particularly given the lack of quantitative assessments at post-mortem examinations. Bone marrow fat (BMF) is the final fat reserve to be mobilised for energy by a calorie-deprived animal during a state of emaciation. Percentage of BMF has been used to study starvation in several species and may provide an objective measure of ante-mortem body condition. This paper reviews the literature on the use of BMF analysis as a post-mortem diagnostic test for ante-mortem starvation. Beginning with a general overview of starvation and usual methods of assessment to describe animals in poor condition, the analysis of BMF is then introduced. Various methods of BMF analysis are discussed, as well as factors that influence the amount of BMF. This review also discusses the limitations of BMF analysis and makes suggestions where future research should be primarily focused.

PLAG1 expression and target genes in the hypothalamo-pituitary system in male mice.

Knockout of pleomorphic adenoma gene 1 (PLAG1) in mice results in reduced fertility. To investigate whether PLAG1 is involved in reproductive control by the hypothalamo-pituitary system in males, we determined PLAG1 expression sites and compared gene expression between hypothalami and pituitary glands from Plag1 knockout and wildtype animals. Abundant expression of PLAG1 was detected throughout the pituitary gland, including gonadotropes and somatotropes. The hypothalamus also contained a large number of PLAG1-expressing cells. PLAG1 was expressed in some gonadotropin-releasing hormone neurons, but not in kisspeptin neurons. Gene ontology analysis indicated upregulation of cell proliferation in both structures, and of cholesterol biosynthesis in the hypothalamus, but functional confirmation is required. Expression levels of pituitary gonadotropins and gonadotropin-releasing hormone receptor, and of brain gonadotropin-releasing hormone and kisspeptin mRNA were unaffected in knockout mice. We conclude that PLAG1 deficiency does not have a major impact on the reproductive control by the hypothalamo-pituitary system.

Forever young: Endocrinology of paedomorphosis in the Mexican axolotl (Ambystoma mexicanum).

The Mexican axolotl (Ambystoma mexicanum) is a salamander species that does not undergo metamorphosis, resulting in the retention of juvenile characteristics in the mature breeding stage (paedomorphosis). Here we review the endocrinological studies investigating the proximate cause of axolotl paedomorphosis with a focus on the hypothalamo-pituitary-thyroid (HPT) axis. It is well established that axolotl paedomorphosis is a consequence of low activity of the HPT axis. The pituitary hormone thyrotropin (TSH) is capable of inducing metamorphosis in the axolotl, which indicates that all processes and interactions in the HPT axis below the pituitary level are functional, but that TSH release is impaired. In metamorphosing species, TSH secretion is largely controlled by the hypothalamic neuropeptide corticotropin-releasing hormone (CRH), which seems to have lost its thyrotropic activity in the axolotl. However, preliminary experiments have not yet confirmed a role for faulty CRH signalling in axolotl paedomorphosis. Other hypothalamic factors and potential pituitary inhibitors need to be investigated to identify their roles in amphibian metamorphosis and axolotl paedomorphosis.

Thyrotropic activity of corticotropin-releasing hormone in an altricial bird species, the zebra finch (Taeniopygia guttata).

In chicken, corticotropin-releasing hormone (CRH) acts as a thyrotropin (TSH)-releasing factor, mediated by the type 2 CRH receptor (CRHR2) on the thyrotropes of the pituitary gland. It is not known whether CRH also controls TSH release in non-precocial avian species that have a different pattern of thyroidal activity during their life cycle. Therefore, we investigated the TSH-releasing capacity of CRH in an altricial species, the zebra finch (Taeniopygia guttata). Cellular localisation of type 1 CRH receptor (CRHR1) and CRHR2 mRNA in the pituitary was determined by in situ hybridisation, combined with immunohistochemical staining of pituitary thyrotropes. In addition, isolated pituitary glands were stimulated with CRH to determine the effect on TSH release. Lastly, the mRNA levels of hormones and receptors involved in the control of thyroidal and adrenal function were measured by qPCR in zebra finch chicks between hatching and fledging, and in adults. Most of the hypophyseal CRHR2 mRNA co-localised with thyrotropes, whereas CRHR1 mRNA was found inbetween thyrotropes. Pituitary glands stimulated in vitro with CRH showed increased secretion of TSH-like activity. Pituitary CRHR2 mRNA expression decreased while pituitary TSHB mRNA and brain CRH mRNA levels increased towards fledging, similar as seen in chicken hatching. These results suggest that CRHR2 expressed on thyrotropes is likely mediating CRH-induced TSH release in altricial avian species like it does in precocial species, and that the increased thyroid hormone levels towards fledging in altricial birds are the result of increased hypothalamic stimulation, in which the thyrotropic activity of CRH may initially play a role.

High seroprevalance of Neospora caninum in dogs in Victoria, Australia, compared to 20 years ago.

BACKGROUND: Canids are definitive hosts of the apicomplexan parasite Neospora caninum, the leading cause of abortion in cattle worldwide. For horizontal transmission from canids to occur, oocysts of N. caninum must be shed by the definitive host into the environment of susceptible intermediate hosts such as cattle. The purpose of this study was to determine the prevalence of N. caninum in canids in Victoria, Australia's leading dairy producing state. RESULTS: Neospora-like oocysts were observed in 8% (18/234) of faecal samples from wild dogs, domestic dogs and red foxes from Victoria, Australia. However, none tested positive for N. caninum DNA using a quantitative PCR. In a separate sample population, blood sera from 483 domestic dogs were tested for anti-N. caninum antibodies using competitive ELISA. A subset of cELISA samples were re-tested using indirect fluorescence antibody test (IFAT). A seroprevalence of 29.8% (144/483; 95% CI: 11.7-47.8%) was calculated when using cELISA; whereas it was 32.9% (27/80; 95% CI: 15.8-51.8%) using IFAT. Potential risk factors were evaluated using univariable analyses and then assessed in separate multivariable models. Using 'aged' dogs as a reference, the seroprevalence of 'adolescent' and 'adult' dogs was 88% (P = 0.05) and 91% (P = 0.08), respectively, indicating seroprevalence increases with age. There was a 19% higher likelihood of infection in rural locations (P = 0.10) relative to urban areas. Jack Russell Terriers had a 22% higher risk of a cELISA-positive result (P = 0.05) regardless of geographical location, age or sex. CONCLUSION: These results demonstrate that exposure to N. caninum in domestic dogs is widespread in Victoria, although faecal oocyst shedding is infrequent. Our results indicate increased N. caninum seroprevalance status in dogs over the past two decades. The results imply that dogs get either exposed to the infected meat more frequently or that vertical dam to foetus transmission is more frequent than previously thought. Our study calls for re-evaluation of historical N. caninum seroprevalance studies, because the attitude to dog diet changes.

PLAG1 deficiency impairs spermatogenesis and sperm motility in mice.

Deficiency in pleomorphic adenoma gene 1 (PLAG1) leads to reduced fertility in male mice, but the mechanism by which PLAG1 contributes to reproduction is unknown. To investigate the involvement of PLAG1 in testicular function, we determined (i) the spatial distribution of PLAG1 in the testis using X-gal staining; (ii) transcriptomic consequences of PLAG1 deficiency in knock-out and heterozygous mice compared to wild-type mice using RNA-seq; and (iii) morphological and functional consequences of PLAG1 deficiency by determining testicular histology, daily sperm production and sperm motility in knock-out and wild-type mice. PLAG1 was sparsely expressed in germ cells and in Sertoli cells. Genes known to be involved in spermatogenesis were downregulated in the testes of knock-out mice, as well as Hsd17b3, which encodes a key enzyme in androgen biosynthesis. In the absence of Plag1, a number of genes involved in immune processes and epididymis-specific genes were upregulated in the testes. Finally, loss of PLAG1 resulted in significantly lowered daily sperm production, in reduced sperm motility, and in several animals, in sloughing of the germinal epithelium. Our results demonstrate that the subfertility seen in male PLAG1-deficient mice is, at least in part, the result of significantly reduced sperm output and sperm motility.

Molecular cloning and tissue distribution of Crh and Pomc mRNA in the fat-tailed dunnart (Sminthopsis crassicaudata), an Australian marsupial.

Like all vertebrates, marsupials respond to stressors with the activation of the hypothalamo-pituitary-adrenal axis. However, peptides operating at the higher regulatory levels of this hormonal system, i.e. corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH), have not been investigated in marsupials. Here we report the molecular cloning of the precursor cDNAs of CRH (prepro-CRH) and of ACTH (proopiomelanocortin; POMC) in an Australian marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata). Dunnart POMC and prepro-CRH are predicted to be peptides of 399 and 200 amino acids, respectively. While the ACTH and ß-endorphin sequences within the POMC sequence are highly conserved, the POMC sequence shows some unique features in this species, and perhaps all Australian marsupials, including the loss of a γ-melanotropin sequence and duplications of the ACTH sequence. Mature dunnart CRH is identical to CRH in human, mouse, rat and chicken. Pomc and Crh mRNA is mainly expressed in dunnart pituitary gland and brain, respectively, but both are also present in a range of peripheral tissues.

Using short-term bioassays to evaluate the endocrine disrupting capacity of the pesticides linuron and fenoxycarb.

Several short-term whole-organism bioassays based on transgenic aquatic models are now under validation by the OECD (Organization for Economic Co-operation and Development) to become standardized test guidelines for the evaluation of the endocrine activity of substances. Evaluation of the endocrine disrupting capacity of pesticides will be a domain of applicability of these future reference tests. The herbicide linuron and the insecticide fenoxycarb are two chemicals commonly used in agricultural practices. While numerous studies indicate that linuron is likely to be an endocrine disruptor, there is little information available on the effect of fenoxycarb on vertebrate endocrine systems. Using whole-organism bioassays based on transgenic Xenopus laevis tadpoles and medaka fry we assessed the potential of fenoxycarb and linuron to disrupt thyroid, androgen and estrogen signaling. In addition we used in silico approach to simulate the affinity of these two pesticides to human hormone receptors. Linuron elicited thyroid hormone-like activity in tadpoles at all concentrations tested and, showed an anti-estrogenic activity in medaka at concentrations 2.5mg/L and higher. Our experiments suggest that, in addition to its previously established anti-androgenic action, linuron exhibits thyroid hormone-like responses, as well as acting at the estrogen receptor level to inhibit estrogen signaling. Fenoxycarb on the other hand, did not cause any changes in thyroid, androgen or estrogen signaling at the concentrations tested.

Effect of in ovo injection of corticotropin-releasing hormone on the timing of hatching in broiler chickens.

In chicken embryos, intravenous injection of corticotropin-releasing hormone (CRH) causes the release of both corticosteroids and thyroid hormones. These hormones initiate and enhance the hatching process, raising the possibility that CRH treatment of the late chicken embryo could accelerate hatching and/or decrease the spread of hatching. We performed a series of exploratory tests to investigate whether in ovo delivery methods of CRH other than intravenous injection that are more practical in a commercial setting, affect hatching time in broilers. Corticotropin-releasing hormone was injected into the air cell, albumen, or amniotic fluid of broiler breeder eggs, in the last week of embryonic development. Average incubation duration was significantly decreased by 22 h when 2 µg of CRH was injected into the air cell on embryonic day 18 (E18) of Cobb eggs. Acceleration of hatching (but only by 8 h) was also seen for Ross chicks when CRH was injected daily into the albumen between E10 and E18. However, repeats of both experiments did not show consistent effects of CRH on hatching time; in most experiments performed, CRH did not affect hatching time. We speculate that the effectiveness of CRH uptake via these delivery methods and/or the duration and magnitude of the thyroxine and corticosterone response to CRH is not sufficient to have a substantial effect on hatching time. We therefore conclude that in ovo CRH treatment does not seem a feasible option as a practical tool to increase hatchery productivity or to investigate the effects of CRH agonists and antagonists on hatching.