Mounting behaviour in finishing pigs: Stable individual differences are not due to dominance or stage of sexual development

Every year around 100 million male piglets are castrated in the EU, usually without anaesthesia or post-operative analgesia. This surgical intervention is painful and stressful. Several main players within the pig industry have voluntarily agreed to end the practice of surgical pig castration in the EU by 2018. One alternative to castration is entire male pig production. However, entire males behave differently than castrates, for example, by performing more mounting behaviour, which is suggested to be a welfare problem. The aim of our study was to develop a comprehensive ethogram of different types of mounting and to investigate properties, causes and consequences of mounting behaviour in finishing pigs. The study included 80 entire male and 80 female pigs from two farrowing batches born six weeks apart. Mixed sex and single-sex housing of pigs are both common in pig farming, so to ensure our study was representative, the 160 pigs were assigned to social groups of 20 in three treatments: entire male pigs only (MM, 2 groups, n = 40), entire females only (FF, 2 groups, n = 40) and entire males and females mixed together (MF, 4 groups, n = 80). Measurements took place during the final six weeks before slaughter (between 63.5 and 105.5 kg). Observations of mounting behaviour on 12 days per batch suggested that: (i) males mounted more than females, (ii) within sex, there was no effect of treatment on the amount of mounting (although the statistical power of the study to detect these effects was low), and (iii) there were individual differences in mounting that were stable over time (within sex). Classification of mounting into different categories revealed that sexual mounting was most common overall and in males but only rare in females. Compared to other types of mounting (e.g. caused by crowding or during a fight), sexual mounts lasted longer and provoked more screaming by the recipient. There were no relationships between mounting behaviour on the one hand and dominance rank in food competition tests, the circulating levels of sex hormones (oestradiol, testosterone and progesterone) at the end of the study, the health scores (lameness and scratches) or weight gain on the other hand. The stable individual differences of mounting over time suggest that mounting behaviour is a trait of the individual rather than the appearance of random outbreaks. However, these differences in mounting cannot be explained by dominance behaviour or by differences in sex hormone concentrations that could indicate the onset of puberty. Mounting behaviour and in particular sexual mounting provoked high pitched screaming of the recipients indicating that mounting is a welfare problem. For the welfare assessment of entire male pig production the performance of mounting behaviour should be considered. (C) 2013 Elsevier B.V. All rights reserved.

Why boys will be boys: two pathways of fetal testicular androgen biosynthesis are needed for male sexual differentiation

Human sexual determination is initiated by a cascade of genes that lead to the development of the fetal gonad. Whereas development of the female external genitalia does not require fetal ovarian hormones, male genital development requires the action of testicular testosterone and its more potent derivative dihydrotestosterone (DHT). The "classic" biosynthetic pathway from cholesterol to testosterone in the testis and the subsequent conversion of testosterone to DHT in genital skin is well established. Recently, an alternative pathway leading to DHT has been described in marsupials, but its potential importance to human development is unclear. AKR1C2 is an enzyme that participates in the alternative but not the classic pathway. Using a candidate gene approach, we identified AKR1C2 mutations with sex-limited recessive inheritance in four 46,XY individuals with disordered sexual development (DSD). Analysis of the inheritance of microsatellite markers excluded other candidate loci. Affected individuals had moderate to severe undervirilization at birth; when recreated by site-directed mutagenesis and expressed in bacteria, the mutant AKR1C2 had diminished but not absent catalytic activities. The 46,XY DSD individuals also carry a mutation causing aberrant splicing in AKR1C4, which encodes an enzyme with similar activity. This suggests a mode of inheritance where the severity of the developmental defect depends on the number of mutations in the two genes. An unrelated 46,XY DSD patient carried AKR1C2 mutations on both alleles, confirming the essential role of AKR1C2 and corroborating the hypothesis that both the classic and alternative pathways of testicular androgen biosynthesis are needed for normal human male sexual differentiation.

Anti-Müllerian hormone levels in girls and adolescents with Turner syndrome are related to karyotype, pubertal development and growth hormone treatment

In girls and adolescents with Turner syndrome (TS), is there a correlation between serum AMH levels and karyotype, spontaneous puberty and other biochemical markers of ovarian function, or growth hormone (GH) therapy? SUMMARY ANSWER: Serum anti-Müllerian hormone (AMH) correlates with karyotype, pubertal development, LH, FSH and are measurable in a higher percentage of TS patients under GH therapy. WHAT IS KNOWN ALREADY: Most girls with TS suffer from incomplete sexual development, premature ovarian failure and infertility due to abnormal ovarian folliculogenesis. Serum AMH levels reflect the ovarian reserve in females, even in childhood. STUDY DESIGN, SIZE, DURATION: Cross-sectional study investigating 270 karyotype proven TS patients aged 0-20 years between 2009 and 2010. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Studies were conducted at three University Children's hospitals in Europe. Main outcome measures were clinical data concerning pubertal development as well as laboratory data including karyotype, serum AMH, LH, FSH, estradiol (E2), inhibin B and IGF. RESULTS AND THE ROLE OF CHANCE: Serum AMH was detectable in 21.9% of all TS girls and correlated strongly with karyotypes. A measurable serum AMH was found in 77% of TS girls with karyotype 45,X/46,XX, in 25% with 'other' karyotypes and in only 10% of 45,X TS girls. A strong relationship was also observed for measurable serum AMH and signs of spontaneous puberty such as breast development [adjusted odds ratio (OR) 19.3; 95% CI 2.1-175.6; P = 0.009] and menarche (crude OR 47.6; 95% CI 4.8-472.9; P = 0.001). Serum AMH correlated negatively with FSH and LH, but did not correlate with E2 and inhibin B. GH therapy increased the odds of having measurable AMH in TS (adjusted OR 4.1; 95% CI 1.9-8.8; P < 0.001). LIMITATIONS, REASONS FOR CAUTION: The cross-sectional design of the study does not allow longitudinal interpretation of the data; for that further studies are needed. High percentage of non-measurable AMH levels in the cohort of TS require categorized analysis. WIDER IMPLICATIONS OF THE FINDINGS: Serum AMH levels are a useful marker of the follicle pool and thus ovarian function in pediatric patients with TS. These findings are in line with the published literature. The finding that GH therapy may affect AMH levels is novel, but must be confirmed by future longitudinal studies.

Of marsupials and men: "Backdoor" dihydrotestosterone synthesis in male sexual differentiation

Following development of the fetal bipotential gonad into a testis, male genital differentiation requires testicular androgens. Fetal Leydig cells produce testosterone that is converted to dihydrotestosterone in genital skin, resulting in labio-scrotal fusion. An alternative 'backdoor' pathway of dihydrotestosterone synthesis that bypasses testosterone has been described in marsupials, but its relevance to human biology has been uncertain. The classic and backdoor pathways share many enzymes, but a 3α-reductase, AKR1C2, is unique to the backdoor pathway. Human AKR1C2 mutations cause disordered sexual differentiation, lending weight to the idea that both pathways are required for normal human male genital development. These observations indicate that fetal dihydrotestosterone acts both as a hormone and as a paracrine factor, substantially revising the classic paradigm for fetal male sexual development.

Ten novel mutations in the NR5A1 gene cause disordered sex development in 46,XY and ovarian insufficiency in 46,XX individuals

Steroidogenic factor-1 (SF-1/NR5A1) is a nuclear receptor that regulates adrenal and reproductive development and function. NR5A1 mutations have been detected in 46,XY individuals with disorders of sexual development (DSD) but apparently normal adrenal function and in 46,XX women with normal sexual development yet primary ovarian insufficiency (POI).

Ethinylestradiol differentially interferes with IGF-I in liver and extrahepatic sites during development of male and female bony fish

Growth and sexual development are closely interlinked in fish; however, no reports exist on potential effects of estrogen on the GH/IGF-I-axis in developing fish. We investigate whether estrogen exposure during early development affects growth and the IGF-I system, both at the systemic and tissue level. Tilapia were fed from 10 to 40 days post fertilization (DPF) with 17alpha-ethinylestradiol (EE(2)). At 50, 75, 90, and 165 DPF, length, weight, sex ratio, serum IGF-I (RIA), pituitary GH mRNA and IGF-I, and estrogen receptor alpha (ERalpha) mRNA in liver, gonads, brain, and gills (real-time PCR) were determined and the results correlated to those of in situ hybridization for IGF-I. Developmental exposure to EE(2) had persistent effects on sex ratio and growth. Serum IGF-I, hepatic IGF-I mRNA, and the number of IGF-I mRNA-containing hepatocytes were significantly decreased at 75 DPF, while liver ERalpha mRNA was significantly induced. At 75 DPF, a transient decline of IGF-I mRNA and a largely reduced number of IGF-I mRNA-containing neurons were observed in the female brain. In both sexes, pituitary GH mRNA was significantly suppressed. A transient downregulation of IGF-I mRNA occurred in ovaries (75 DPF) and testes (90 DPF). In agreement, in situ hybridization revealed less IGF-I mRNA signals in granulosa and germ cells. Our results show for the first time that developmental estrogen treatment impairs GH/IGF-I expression in fish, and that the effects persist. These long-lasting effects both seem to be exerted indirectly via inhibition of pituitary GH and directly by suppression of local IGF-I in organ-specific cells.

Social Factors Associated with Readiness for Sexual Activity in Adolescents: A Population-Based Cohort Study

Various factors are associated with sexual activity in adolescence and it is important to identify those that promote healthy and adaptive romantic and sexual development. The objectives of this study were to describe rates of early sexual intercourse (before 16 years) and sexual readiness in adolescence and to assess the extent to which these were social patterned. We prospectively studied nearly 5,000 15-year-olds from the Avon Longitudinal Study of Parents and Children, a UK birth cohort. Between 2006 and 2008, female and male participants answered a computer assisted interview about romantic and sexual behaviors in the last year. Predictors of sexual intercourse and readiness for sexual intercourse were examined across a range of sociodemographic measures. Overall, 17.7 % (95 % CI 16.7 %, 18.9 %) of participants reported having had sexual intercourse in the last year, with more girls than boys reporting sexual experience (risk ratio 1.30, 95 % CI 1.15, 1.47). Of these, one-third of both male and female were classed as unready because they were unwilling, lacking in autonomy, felt regret or had not used contraception. There was strong evidence of social patterning for sexual activity with higher rates for young people from poorer homes, with lower social class, and with younger, less educated mothers. In contrast, among 860 young people who had had sexual intercourse, there was no clear evidence of associations between social factors and sexual readiness. The lack of social patterning in sexual readiness supports the provision of comprehensive education to develop life skills for adolescents across all social groups.

Clinical and biochemical consequences of p450 oxidoreductase deficiency

Patients with P450 oxidoreductase (POR) deficiency typically present with adrenal insufficiency, genital anomalies and bony malformations resembling the Antley-Bixler craniosynostosis syndrome. Since our first report in 2004, more than 40 POR mutations have been identified in over 65 patients. POR is the obligate electron donor to all microsomal P450 enzymes, including the steroidogenic enzymes CYP17A1, CYP21A2 and CYP19A1. POR deficiency may cause disordered sexual development manifested as genital undervirilization in 46, XY newborns as well as overvirilization in those who are 46, XX. This may be explained by impaired aromatization of fetal androgens that may cause maternal virilization and low urinary estriol levels during pregnancy. In addition, the alternate 'backdoor' pathway of androgen biosynthesis, which leads to dihydrotestosterone production bypassing androstenedione and testosterone, may also play a role. Functional assays studying the effects of POR mutations on steroidogenesis showed that several POR variants impaired CYP17A1, CYP21A2 and CYP19A1 activities to different degrees, indicating that each POR variant must be studied separately for each potential target P450 enzyme. POR variants may also affect skeletal development and drug metabolism. As most drugs are metabolized by hepatic microsomal P450 enzymes, studies of the impact of POR mutations on drug-metabolizing P450s are particularly important.

Deletion of P399_E401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency

P450 oxidoreductase (POR) is the electron donor for all microsomal P450s including steroidogenic enzymes CYP17A1, CYP19A1 and CYP21A2. We found a novel POR mutation P399_E401del in two unrelated Turkish patients with 46,XX disorder of sexual development. Recombinant POR proteins were produced in yeast and tested for their ability to support steroid metabolizing P450 activities. In comparison to wild-type POR, the P399_E401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17α-hydroxylation of progesterone by 76%, 17,20-lyase action on 17OH-pregnenolone by 69%, aromatization of androstenedione by 85% and cytochrome c reduction activity by 80%. Protein structure analysis of the three amino acid deletion P399_E401 revealed reduced stability and flexibility of the mutant. In conclusion, P399_E401del is a novel mutation in POR that provides valuable genotype-phenotype and structure-function correlation for mutations in a different region of POR compared to previous studies. Characterization of P399_E401del provides further insight into specificity of different P450s for interaction with POR as well as nature of metabolic disruptions caused by more pronounced effect on specific P450s like CYP17A1 and aromatase.

Concentration- and time-dependent effects of the synthetic estrogen, 17alpha-ethinylestradiol, on reproductive capabilities of the zebrafish, Danio rerio

Partial or full life-cycle tests are needed to assess the potential of endocrine-disrupting compounds (EDCs) to adversely affect development and reproduction of fish. Small fish species such as zebrafish, Danio rerio, are under consideration as model organisms for appropriate test protocols. The present study examines how reproductive effects resulting from exposure of zebrafish to the synthetic estrogen 17alpha-ethinylestradiol (EE2) vary with concentration (0.05 to 10 ng EE2 L(-1), nominal), and with timing/duration of exposure (partial life-cycle, full life-cycle, and two-generation exposure). Partial life-cycle exposure of the parental (F1) generation until completion of gonad differentiation (0-75 d postfertilization, dpf) impaired juvenile growth, time to sexual maturity, adult fecundity (egg production/female/day), and adult fertilization success at 1.1 ng EE2 L(-1) and higher. Lifelong exposure of the F1 generation until 177 dpf resulted in lowest observed effect concentrations (LOECs) for time to sexual maturity, fecundity, and fertilization success identical to those of the developmental test (0-75 dpf), but the slope of the concentration-response curve was steeper. Reproduction of zebrafish was completely inhibited at 9.3 ng EE2 L(-1), and this was essentially irreversible as a 3-mo depuration restored fertilization success to only a very low rate. Accordingly, elevated endogenous vitellogenin (VTG) synthesis and degenerative changes in gonad morphology persisted in depurated zebrafish. Full life-cycle exposure of the filial (F2) generation until 162 dpf impaired growth, delayed onset of spawning and reduced fecundity and fertilization success at 2.0 ng EE2 L(-1). In conclusion, results show that the impact of estrogenic agents on zebrafish sexual development and reproductive functions as well as the reversibility of effects, varies with exposure concentration (reversibility at < or = 1.1 ng EE2 L(-1) and irreversibility at 9.3 ng EE2 L(-1)), and between partial and full life-cycle exposure (exposure to 10 ng EE2 L(-1) during critical period exerted no permanent effect on sexual differentiation, but life-cycle exposure did).

P450 oxidoreductase deficiency - a new form of congenital adrenal hyperplasia

Patients with adrenal insufficiency, genital anomalies and bony malformations resembling the Antley- Bixler syndrome (a craniosynostosis syndrome), are likely to have P450 oxidoreductase (POR) deficiency. Since our first report in 2004, about 26 recessive POR mutations have been identified in 50 patients. POR is the obligate electron donor to all microsomal (type II) P450 enzymes, including the steroidogenic enzymes CYP17A1, CYP21A2 and CYP19A1. POR deficiency may cause disordered sexual development manifested as genital undervirilization in 46,XY newborns as well as overvirilization in those who are 46,XX. This may be explained by impaired aromatization of fetal androgens which may also lead to maternal virilization and low urinary estriol levels during pregnancy. A role for the alternate 'backdoor' pathway of androgen biosynthesis, leading to dihydrotestosterone production bypassing androstenedione and testosterone, has been suggested in POR deficiency but remains unclear. POR variants may play an important role in drug metabolism, as most drugs are metabolized by hepatic microsomal P450 enzymes. However, functional assays studying the effects of specific POR mutations on steroidogenesis showed that several POR variants impaired CYP17A1, CYP21A2 and CYP19A1 activities to different degrees, indicating that each POR variant must be studied separately for each potential target P450 enzyme. Thus, the impact of POR mutations on drug metabolism by hepatic P450s requires further investigation.

Infection of primary canine duodenal epithelial cell cultures with Neospora caninum

According to current knowledge, sexual development of the apicomplexan parasite Neospora caninum takes place in the canine intestine. However, to date there is no information on the interaction between the parasite and the canine intestinal epithelium, and, next to the clinical and in vivo research tools, an in vitro model comprised of canine intestinal cells infected with N. caninum would be very helpful for investigations at the cellular level. Following the isolation of cells of neonatal canine duodenum and growth of cell cultures to monolayers for 5-6 days, canine intestinal epithelial cells were exposed to cell culture-derived N. caninum tachyzoites and bradyzoites. The host cells remained viable during in vitro culture for an average of 2 wk. During this time span, N. caninum was found to readily adhere to any surface area of these cells, but infection took mostly place at sites where microvilli-like structures were missing, e.g., at the cell periphery, with tachyzoites exhibiting at least 3-4 times increased invasive capacities compared to bradyzoites. Once intracellular, parasites resided within a parasitophorous vacuole, moved toward the vicinity of the nucleus and the more distal portion of the epithelial cells, and proliferated to form vacuoles of not more than 2-4 parasites, which were surrounded by numerous mitochondria. Immunofluorescence staining and TEM of infected cells showed that the expression of cytokeratins and the structural integrity of desmosomes and tight junctions were not notably altered during infection. Furthermore, no changes could be detected in the alkaline phosphatase activities in cell culture supernatants of infected and noninfected cells. Canine duodenal epithelial cell cultures represent a useful tool for future studies on the characteristics of the intestinal phases of N. caninum infection.

Steroidogenesis of the testis - new genes and pathways

Defects of androgen biosynthesis cause 46,XY disorder of sexual development (DSD). All steroids are produced from cholesterol and the early steps of steroidogenesis are common to mineralocorticoid, glucocorticoid and sex steroid production. Genetic mutations in enzymes and proteins supporting the early biosynthesis pathways cause adrenal insufficiency (AI), DSD and gonadal insufficiency. The classic androgen biosynthesis defects with AI are lipoid CAH, CYP11A1 and HSD3B2 deficiencies. Deficiency of CYP17A1 rarely causes AI, and HSD17B3 or SRD5A2 deficiencies only cause 46,XY DSD and gonadal insufficiency. All androgen biosynthesis depends on 17,20 lyase activity of CYP17A1 which is supported by P450 oxidoreductase (POR) and cytochrome b5 (CYB5). Therefore 46,XY DSD with apparent 17,20 lyase deficiency may be due to mutations in CYP17A1, POR or CYB5. Illustrated by patients harboring mutations in SRD5A2, normal development of the male external genitalia depends largely on dihydrotestosterone (DHT) which is converted from circulating testicular testosterone (T) through SRD5A2 in the genital skin. In the classic androgen biosynthetic pathway, T is produced from DHEA and androstenedione/-diol in the testis. However, recently found mutations in AKR1C2/4 genes in undervirilized 46,XY individuals have established a role for a novel, alternative, backdoor pathway for fetal testicular DHT synthesis. In this pathway, which has been first elucidated for the tammar wallaby pouch young, 17-hydroxyprogesterone is converted directly to DHT by 5α-3α reductive steps without going through the androgens of the classic pathway. Enzymes AKR1C2/4 catalyse the critical 3αHSD reductive reaction which feeds 17OH-DHP into the backdoor pathway. In conclusion, androgen production in the fetal testis seems to utilize two pathways but their exact interplay remains to be elucidated.

Pharmacogenomics of human P450 oxidoreductase

Cytochrome P450 oxidoreductase (POR) supports reactions of microsomal cytochrome P450 which metabolize drugs and steroid hormones. Mutations in POR cause disorders of sexual development. P450 oxidoreductase deficiency (PORD) was initially identified in patients with Antley-Bixler syndrome (ABS) but now it has been established as a separate disorder of sexual development (DSD). Here we are summarizing the work on variations in POR related to metabolism of drugs and xenobiotics. We have compiled mutation data on reported cases of PORD from clinical studies. Mutations found in patients with defective steroid profiles impact metabolism of steroid hormones as well as drugs. Some trends are emerging that establish certain founder mutations in distinct populations, with Japanese (R457H), Caucasian (A287P), and Turkish (399-401) populations showing repeated findings of similar mutations. Most other mutations are found as single occurrences. A large number of different variants in POR gene with more than 130 amino acid changes are now listed in databases. Among the polymorphisms, the A503V is found in about 30% of all alleles but there are some differences across different population groups.