Defining potential targets of prenatal androgen excess: Expression analysis of androgen receptor on hypothalamic neurons in the fetal female mouse brain.

Polycystic ovary syndrome (PCOS) is a female endocrine disorder that is associated with prenatal exposure to excess androgens. In prenatally androgenized (PNA) mice that model PCOS, GABAergic neural transmission to and innervation of GnRH neurons is increased. Evidence suggests that elevated GABAergic innervation originates in the arcuate nucleus (ARC). We hypothesized that GABA-GnRH circuit abnormalities are a direct consequence of PNA, resulting from DHT binding to androgen receptor (AR) in the prenatal brain. However, whether prenatal ARC neurons express AR at the time of PNA treatment is presently unknown. We used RNAScope in situ hybridization to localize AR mRNA (Ar)-expressing cells in healthy gestational day (GD) 17.5 female mouse brains and to assess coexpression levels in specific neuronal phenotypes. Our study revealed that less than 10% of ARC GABA cells expressed Ar. In contrast, we found that ARC kisspeptin neurons, critical regulators of GnRH neurons, were highly colocalized with Ar. Approximately 75% of ARC Kiss1-expressing cells also expressed Ar at GD17.5, suggesting that ARC kisspeptin neurons are potential targets of PNA. Investigating other neuronal populations in the ARC we found that ~50% of pro-opiomelanocortin (Pomc) cells, 22% of tyrosine hydroxylase (Th) cells, 8% of agouti-related protein (Agrp) cells and 8% of somatostatin (Sst) cells express Ar. Lastly, RNAscope in coronal sections showed Ar expression in the medial preoptic area (mPOA), and the ventral part of the lateral septum (vLS). These Ar-expressing regions were highly GABAergic, and 22% of GABA cells in the mPOA and 25% of GABA cells in the vLS also expressed Ar. Our findings identify specific neuronal phenotypes in the ARC, mPOA, and vLS that are androgen sensitive in late gestation. PNA-induced functional changes in these neurons may be related to the development of impaired central mechanisms associated with PCOS-like features.

Deletion of Androgen Receptors From Kisspeptin Neurons Prevents PCOS Features in a Letrozole Mouse Model.

Polycystic ovarian syndrome (PCOS) is the leading cause of anovulatory infertility and is a heterogenous condition associated with a range of reproductive and metabolic impairments. While its etiology remains unclear, hyperandrogenism and impaired steroid negative feedback have been identified as key factors underpinning the development of PCOS-like features both clinically and in animal models. We tested the hypothesis that androgen signaling in kisspeptin-expressing neurons, which are key drivers of the neuroendocrine reproductive axis, is critically involved in PCOS pathogenesis. To this end, we used a previously validated letrozole (LET)-induced hyperandrogenic mouse model of PCOS in conjunction with Cre-lox technology to generate female mice exhibiting kisspeptin-specific deletion of androgen receptor (KARKO mice) to test whether LET-treated KARKO females are protected from the development of reproductive and metabolic PCOS-like features. LET-treated mice exhibited hyperandrogenism, and KARKO mice exhibited a significant reduction in the coexpression of kisspeptin and androgen receptor mRNA compared to controls. In support of our hypothesis, LET-treated KARKO mice exhibited improved estrous cyclicity, ovarian morphology, and insulin sensitivity in comparison to LET-treated control females. However, KARKO mice were not fully protected from the effects of LET-induced hyperandrogenism and still exhibited reduced corpora lutea numbers and increased body weight gain. These data indicate that increased androgen signaling in kisspeptin-expressing neurons plays a critical role in PCOS pathogenesis but highlight that other mechanisms are also involved.

Prenatal androgenization causes expression changes of progesterone and androgen receptor mRNAs in the arcuate nucleus of female mice across development.

Prenatal exposure to excess androgens is associated with the development of polycystic ovary syndrome (PCOS). In prenatally androgenised (PNA) mice, a model of PCOS, progesterone receptor (PR) protein expression is reduced in arcuate nucleus (ARC) GABA neurons. This suggests a mechanism for PCOS-related impaired steroid hormone feedback and implicates androgen excess with respect to inducing transcriptional repression of the PR-encoding gene Pgr in the ARC. However, the androgen sensitivity of ARC neurons and the relative gene expression of PRs over development and following prenatal androgen exposure remain unknown. Here, we used a quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) of microdissected ARC to determine the relative androgen receptor (Ar) and progesterone receptor (Pgr) gene expression in PNA and control mice at five developmental timepoints. In a two-way analysis of variance, none of the genes examined showed expression changes with a statistically significant interaction between treatment and age, although PgrA showed a borderline interaction. For all genes, there was a statistically significant main effect of age on expression levels, reflecting a general increase in expression with increasing age, regardless of treatment. For PgrB and Ar, there was a statistically significant main effect of treatment, indicating a change in expression following PNA (increased for PgrB and decreased for Ar), regardless of age. For PgrA, there was a borderline main effect of treatment, suggesting a possible change in expression following PNA, regardless of age. PgrAB gene expression changes showed no significant main effect of treatment. We additionally examined androgen and progesterone responsiveness specifically in P60 ARC GABA neurons using RNAScope® (Advanced Cell Diagnostics, Inc.) in situ hybridization. This analysis revealed that Pgr and Ar were expressed in the majority of ARC GABA neurons in normal adult females. However, our RNAScope® analysis did not show significant changes in Pgr or Ar expression within ARC GABA neurons following PNA. Lastly, because GABA drive to gonadotropin-releasing hormone neurons is increased in PNA, we hypothesised that PNA mice would show increased expression of glutamic acid decarboxylase (GAD), the rate-limiting enzyme in GABA production. However, the RT-qPCR showed that the expression of GAD encoding genes (Gad1 and Gad2) was unchanged in adult PNA mice compared to controls. Our findings indicate that PNA treatment can impact Pgr and Ar mRNA expression in adulthood. This may reflect altered circulating steroid hormones in PNA mice or PNA-induced epigenetic changes in the regulation of Pgr and Ar gene expression in ARC neurons.

Fungus-originated glucanase and monooxygenase genes in creeping bent grass (Agrostis stolonifera L.).

Recent studies have revealed presence of fungus-originated genes in genomes of cool-season grasses, suggesting occurrence of multiple ancestral gene transfer events between the two distant lineages. The current article describes identification of glucanase-like and monooxygenase-like genes from creeping bent grass, as lateral gene transfer candidates. An in silico analysis suggested presence of the glucanase-like gene in Agrostis, Deyeuxia, and Polypogon genera, but not in other species belonging to the clade 1 of the Poeae tribe. Similarly, the monooxygenase-like gene was confined to Agrostis and Deyeuxia genera. A consistent result was obtained from PCR-based screening. The glucanase-like gene was revealed to be ubiquitously expressed in young seedlings of creeping bent grass. Although expression of the monooxygenase-like gene was suggested in plant tissues, the levels were considerably lower than those of the glucanase-like gene. A phylogenetic analysis revealed close relationships of the two genes between the corresponding genes in fungal endophyte species of the Epichloë genus, suggesting that the genes originated from the Epichloë lineage.

Development of a biosecurity assessment tool and the assessment of biosecurity levels by this tool on Japanese commercial swine farms.

It is well known that infectious diseases such as porcine reproductive and respiratory syndrome (PRRS) and porcine epidemic diarrhea (PED) decrease herd productivity and lead to economic loss. It is believed that biosecurity practices are effective for the prevention and control of such infectious diseases. Therefore, the objective of the present study was to investigate whether or not an association between biosecurity level and herd productivity, as well as disease status exists on Japanese commercial swine farms. The present study was conducted on 141 farms. Biosecurity in each farm was assessed by a biosecurity assessment tool named BioAsseT. BioAsseT has a full score of 100 and consists of three sections (external biosecurity, internal biosecurity and diagnostic monitoring). Production data for number of pigs weaned per sow per year (PWSY) and post-weaning mortality per year (PWM) were collected for data analysis. Regarding PRRS status, the farms were categorized into two groups: unknown or unstable and stable or negative. In addition, these farms were categorized based on their PED status, either positive or negative. The total BioAsseT score was associated with herd productivity: as total score increased by 1, PWSY increased by 0.104 pigs and PWM decreased by 0.051 % (P < 0.05). Herd productivity was associated with the score of external and internal biosecurity (P < 0.05), but did not correlate with the score of diagnostic monitoring. Regarding PRRS status, farms with an unknown or unstable status had lower total score than those with stable or negative status (P < 0.05). Similarly, PED positive farms had a lower total score compared to PED negative farms (P < 0.05). In conclusion, the present study provides evidence for the association between high biosecurity levels and increased herd productivity as well as a decreased risk for novel introductions of infectious diseases such as PED.

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.

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.

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.

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.

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.

Impact of a porcine epidemic diarrhea outbreak on swine productivity in Japan: a retrospective cohort study.

The objective was to investigate porcine epidemic diarrhea (PED) outbreak that occurred in 2014 in Japan and its effects on herd-level productivity using a data recording system (PigINFO). The study herds were selected from farrow-to-finish herds (n=99) that entered in the PigINFO system between July 2013 and March 2015. From 1 April to 30 June 2014 (PED epidemic), any herds with clinical signs of PED and feces positive for porcine epidemic diarrhea virus (PEDV) on polymerase chain reaction analysis and/or immunohistochemical staining were defined as PED-positive (n=38). They were further classified into those with long PED periods (L-PED-positive; n=28) and those with short PED periods (S-PED-positive; n=10). Herds with no clinical signs of PED were classified as PED-negative (n=61). Herd-level production data, including preweaning mortality (%; PRWM), postweaning mortality (%; POWM), pigs weaned per litter (PWL), pigs born alive per litter, litters per mated female per year and pigs marketed per sow (MP), were calculated every 3 months during study period. During the PED epidemic, L-PED-positive herds had significantly higher PRWM and POWM than PED-negative herds, and L-PED-positive and S-PED-positive herds had significantly lower PWL. During October-December 2014, L-PED-positive herds had significantly fewer MP than PED-negative herds. The PED outbreak increased mortality and consequently reduced the numbers of marketed pigs. The rapid control of an outbreak is important for reducing the financial losses arising from PED infections.

Corticotropin-releasing hormone: Mediator of vertebrate life stage transitions?

Hormones, particularly thyroid hormones and corticosteroids, play critical roles in vertebrate life stage transitions such as amphibian metamorphosis, hatching in precocial birds, and smoltification in salmonids. Since they synergistically regulate several metabolic and developmental processes that accompany vertebrate life stage transitions, the existence of extensive cross-communication between the adrenal/interrenal and thyroidal axes is not surprising. Synergies of corticosteroids and thyroid hormones are based on effects at the level of tissue hormone sensitivity and gene regulation. In addition, in representative nonmammalian vertebrates, corticotropin-releasing hormone (CRH) stimulates hypophyseal thyrotropin secretion, and thus functions as a common regulator of both the adrenal/interrenal and thyroidal axes to release corticosteroids and thyroid hormones. The dual function of CRH has been speculated to control or affect the timing of vertebrate life history transitions across taxa. After a brief overview of recent insights in the molecular mechanisms behind the synergic actions of thyroid hormones and corticosteroids during life stage transitions, this review examines the evidence for a possible role of CRH in controlling vertebrate life stage transitions.