Abstract. Introduction. RBMOnline - Vol 8. No Reproductive BioMedicine Online; on web 10 February 2004

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1 RBMOnline - Vol 8. No Reproductive BioMedicine Online; on web 10 February 2004 Article The Booroola (FecB) mutation is associated with smaller adrenal glands in young adult ewes Carlos JH Souza is currently a research scientist at EMBRAPA Genetic Resources and Biotechnology, Brasilia, Brazil. He obtained his BVetSc and MSc degrees in Animal Reproduction from the Faculty of Veterinary Medicine of the Federal University of Rio Grande do Sul, Brazil. These qualifications were followed by a PhD from the Faculty of Medicine of the University of Edinburgh, Scotland. He held post-doctoral positions at EMBRAPA in Brazil and the Centre for Reproductive Biology in Edinburgh. His research interests are paracrine control of ovarian follicle growth, genetic regulation of ovulation rate, testicular stem cell transplant and immunocontraception. Dr Carlos JH Souza CJH Souza 1,2, DT Baird 2 1 EMBRAPA Genetic Resource and Biotechnology, Parque Estação Biológica s/n, Final W5 norte, CEP , Brasilia, Brazil 2 University of Edinburgh, Department of Reproductive and Developmental Sciences, Centre for Reproductive Biology, Chancellor s Building, Little France Crescent, Edinburgh EH16 4SB, UK 3 Correspondence: csouza@cenargen.embrapa.br Abstract The Booroola (FecB) phenotype is associated with a mutation in the bone morphogenetic protein (BMP) receptor 1B. The BMP action is important during development; surprisingly the only differences so far observed in adult animals are restricted to the ovaries where precocious development of the antral follicles and increased ovulation rate of mutant ewes is observed. The internal organs of 17 ewes homozygous for the mutation (BB) and 18 wild-type ewes (++) were macroscopically examined and weighed. No macroscopic differences were found, and the weight of the heart, liver, lungs, kidneys, and spleen were similar for both genotypes (P > 0.05). In contrast, the adrenals of BB ewes were lighter than those of ++ ewes (P < 0.05). The effect of the mutation on the adrenal function of cortisol secretion was measured at basal level and after an adrenocorticotrophic hormone challenge, before and after dexamethasone suppression. The Booroola mutation had no effect (P > 0.05) in any of these conditions. These findings indicate that the Booroola mutation also affects the size of the adrenal glands and suggest that the mutated gene could be important in the development of other organs in addition to the ovary. However, in the mutant ewes the function of the adrenal glands is not compromised or it is compensated. Keywords: adrenal, BMP, Booroola, development, ovulation, sheep 414 Introduction The Booroola phenotype (FecB) is characterized by ewes with increased ovulation rate and litter size associated with the precocious development of a large number of antral follicles that are smaller than in the wild type (Baird and Campbell, 1998). This phenotype has been recently associated with a single point mutation in the kinase domain of the gene coding for the bone morphogenetic protein (BMP) receptor 1B (Mulsant et al., 2001; Souza et al., 2001; Wilson et al., 2001). The BMP are very important in key steps of mammalian development as can be inferred from rodent models that are deficient for ligands, receptors and intracellular signalling proteins (for review see Goumans and Mummery, 2000). These effects can be profound as in BMP4-null mice where the deficiency is lethal in early gestation (Lawson et al., 1999), or mild as in the case of mice lacking BMP6 function that are viable and fertile, with minor skeletogenesis delay at late gestation but newborn and adult mutants are indistinguishable from the wild type (Solloway et al., 1998). Mice deficient for the BMP receptor 1B (BMPR1B) are viable and exhibit skeletal defects that are largely restricted to digit formation (Baur et al., 2000; Yi et al., 2000). However the BMPR1B is essential for many aspects of female fertility, with mutant mice exhibiting irregular oestrous cycles, impaired pseudopregnancy response, defects in cumulus cell expansion that prevent fertilization in vivo, decreased aromatase production in granulosa cells and failure in endometrial gland formation (Yi et al., 2001). Sheep fetuses carrying the Booroola mutation have lower body weight at different gestational ages (40, 70, 90 and 135 days)

2 and female fetuses have lighter ovaries and adrenal glands at late gestation (95 days) with the difference in ovarian size persisting to day 135 (Smith et al., 1993). The FecB mutation is also associated with retarded development of the heart (day 28), mesonephros (days 30 40) and ovary (day 30 to early neonatal life). The ovaries of animals homozygous for the mutation (BB) have fewer oogonia (days 30 40), primordial follicles (day 75 90) and growing follicles (day 120 to 6 weeks after birth) (McNatty et al., 1995) than the wild type (++). However, none of these differences in development apart from those of the ovary seems to persist to term (145 days). The action of BMP is important in several organs during development, but surprisingly the only differences so far observed in adult Booroola mutant animals are restricted to the ovary. The aim of this study is to investigate the effects of the Booroola mutation on the development of other internal organs in ewes homozygous for the mutation and in wild-type ewes, and to investigate the effect of the mutation on adrenal function. Materials and methods Organ morphometry A total of 35 Scottish Black Face Merino ewes aged months were used in this study: 17 were homozygous for the Booroola mutation (BB) and 18 were wild-type animals (++) matched by body weight. The animals were subjected to 12 h fasting, and they were weighed before being killed by overdose of pentobarbitone sodium (Euthatal, Rhône Mérieux Ltd, Harlow, Essex, UK). The internal organs were immediately dissected free from connective tissues, examined macroscopically and weighed. In three BB and four ++ ewes (22 months of age) the right adrenal was collected for histological examination. The glands were cut into quarters and fixed overnight in 4% paraformaldehyde in phosphate buffered saline, 0.01 mol/l ph 7.6 (PBS, Sigma, Dorset, UK), and transferred to 70% ethanol until processing. The fixed tissue was embedded in paraffin after the usual dehydration steps. Sections of 5 mm were dewaxed and rehydrated in decreasing concentrations of alcohol (90%, 70%, 30% and distilled water), the sections were stained with haematoxylin and eosin (BDH, UK), dehydrated with increasing concentrations of alcohol and air dried. Slides with a sagittal section of the gland were used to measure the cortical and medullary compartments, and the percentage of each compartment was estimated. The histological appearance of the gland and cell size were evaluated in slides from these animals, by two observers. Adrenal function Twelve Scottish Black Face Merino mature ewes, six BB and six ++ animals, were studied during the anoestrous season. The animals were housed indoors at the Marshall Building, Roslin, Midlothian, Edinburgh, under natural lighting and received a maintenance diet of hay and pelletted ration. The day before the start of blood sampling the jugular vein was cannulated under local anaesthesia as previously described (Souza et al., 1996). The ewes were placed in metabolism crates and received a prophylactic treatment of broad spectrum longacting antibiotic (3 ml i.m.; Clamoxil, SmithKline Beecham, Surrey, UK). Samples of jugular (5 ml) venous blood were collected at 15 min intervals for 45 min before and 120 min after an adrenocorticotrophic hormone (ACTH) challenge, which consisted of intravenous injection of the ACTH analogue, tetracosactrin acetate (Synacthen, Alliance Pharmaceuticals Ltd, Chippenham, Wiltshire, UK). After the first challenge, the animals were injected with dexamethasone every 12 h for 2 days and the ACTH challenge repeated. The blood samples were centrifuged at 4 C immediately after collection, the plasma was separated and stored at 20 C until assay. Cortisol plasma concentrations were measured in duplicate using previously described double-antibody radioimmunoassay after solvent extraction (Hagan and Brooks, 1996). The sensitivity of the assay was 15 pg/ml and the intra and interassay variation were less than 15% in the ED range. Statistical analysis The effects of genotype, age and type of birth on organ weight were analysed by analysis of variance (ANOVA) with Systat software (Systat Inc., Evanston, IL, USA), using total body mass as a covariate. The effects of genotype and dexamethasone treatment on basal cortisol plasma concentration and the concentration after ACTH challenge were analysed by repeated samples ANOVA using the same software. Results There were no macroscopic differences between genotypes in the organs examined. The total body weight (Table 1) and the weights of the heart, liver, lungs, kidneys, and spleen were similar for both genotypes (P > 0.05, Table 1). In contrast, the left adrenal was heavier than the right (P < 0.05, Table 1) and the adrenal glands of BB ewes were significantly lighter than ++ ewes on both sides (P < 0.05, Table 1). Analyses of variance were performed to include other factors that could affect adrenal weight i.e. age and type of birth (singleton or multiple). As there was no interaction between the weight of right or left adrenal glandss and genotype, the weights of both were pooled. The combined adrenal weight was influenced by age and type of birth (P < 0.05) but the interaction of these two variables with genotype was not significant (P>0.05). The adrenals were lighter in animals at 10 months of age than at 22 months (P < 0.05) and in animals born as singletons rather than as multiples (P < 0.05). The variation in size of the adrenals was also investigated using body weight as a covariate; the effect of genotype was maintained (P < 0.05) while the effects of age and type of birth were not significant (P > 0.05). There was no effect of genotype on the percentage of each compartment in the gland (P > 0.05) with the cortical region comprising 68% of the gland while the rest was medulla. There were also no differences that could be detected by observation in the overall architecture of the gland or in cell size for the different genotypes (Figure 1). 415

3 Table 1. Mean weight (± SE) of body and internal organs of ewes homozygous for the Booroola mutation (BB) or wild type (++) at 10 and 22 months of age. Mean weight ++ (10 months) BB (10 months) ++ (22 months) BB (22 months) Body (kg) 27.9 ± ± ± ± 1.4 Heart (g) ± ± ± ± 3.5 Liver (g) ± ± ± ± 5.8 Lung (g) ± ± ± ± 11.2 Left kidney (g) 41.1 ± ± ± ± 1.9 Right kidney (g) 41.1 ± ± ± ± 2.0 Spleen (g) ± ± ± ± 17.9 Left adrenal (g) 1.1 ± 0.06 a 0.9 ± 0.04 a 1.4 ± 0.06 a 1.1 ± 0.09 a Right adrenal (g) 0. 9 ± 0.04 a 0.8 ± 0.03 a 1.3 ± 0.07 a 1.1 ± 0.05 a a Significant difference (P < 0.05) between genotypes. Figure 1. Adrenal glands from ewes homozygous for the Booroola mutation (A and C) and wild-type ewes (B and D), showing the fibrous capsule (ca) and the cortical (co) and medullary (m) compartments of the gland. The squares in the top panels highlight the area enlarged in the bottom panels showing the capsule (ca), zona glomerulosa (g) and the zona fascilucata (f) of the cortical region. Scale bars: 50 µm (A and B); 200 µm (C and D). 416 Cortisol concentrations The concentration of plasma cortisol prior to the injection of ACTH analogue was similar for both genotypes (P > 0.05, Figure 2). After analogue injection, the cortisol concentrations rose (P < 0.05, Figure 2) as expected, but remained similar in both the genotypes (P > 0.05, Figure 2). After dexamethasone treatment the basal concentrations of cortisol were undetectable or markedly reduced (P < 0.05, Figure 2); cortisol concentrations increased after ACTH injection (P < 0.05, Figure 2) but were of a lesser magnitude than previously observed (P < 0.05, Figure 2). The response to the challenge was not affected by the genotype of the ewes (P > 0.05, Figure 2).

4 fundamental to life it may be compensated in the mutant animals. Although only one dose of ACTH was tested there was a five-fold increase in cortisol concentration, which demonstrates that the mutant ewes have adequate adrenal reserves to respond to stress. Figure 2. Mean concentration of cortisol (ng/ml) in ewes homozygous for the Booroola mutation (open symbols) and wild-type ewes (filled symbols), minutes before and after an injection of ACTH analogue. The response is shown after a 48- hour treatment with dexamethasone (circles) and in untreated animals (squares). Discussion The findings of smaller adrenal glands in young adult Booroola ewes expand the observations of the effect of the mutation on the development of other organs apart from those directly involved in reproduction (McNatty et al., 1995), and confirm that the reduction in adrenal size observed during fetal life in mutant animals persists to adulthood. The cortical adrenal cells have the same origin, the mesonephros, (Upadhyay and Zamboni, 1982) as the granulosa cells where the Booroola phenotype is consistently observed. The adrenal medulla is embryologically derived from the neural crest of the ectoderm. The BMP and their receptors in the avian neural crest are involved in the differentiation into the adrenal phenotype (McPherson et al., 2000). Thus it is possible that impairment of function associated with a mutation in BMPR1B would result in abnormalities in the formation of those organs whose development is dependent on BMP signalling. A similar situation seems to arise in the ovaries (Fabre et al., 2003), influencing follicle growth and multiple ovulation via paracrine factors. BMP receptors and putative ligands are ubiquitously expressed within the ovary, and BMP are involved in the paracrine regulation of FSH action. Thus if the Booroola mutation is causing a reduction in BMPR1B signalling, it may act on an inhibitor of follicle differentiation (Souza et al., 2003). The mechanism of increased ovulation rate of the Booroola mutation differs from that observed in women over 30 years of age because is not dependent on the increased FSH levels in the early follicular phase and is not influenced by the age of the animals (Bulnes, Souza, Campbell and Baird, unpublished data). This study showed no difference in the function of the adrenal gland as judged by basal concentration of cortisol or its response to stimulation with ACTH, despite the reduction in organ mass. Ewes immunized against αc inhibin showed an increase in adrenal weight of around 30% when compared with controls, although this increase did not change basal or ACTHstimulated cortisol secretion. (McNeilly et al., 1994). The reduction of 20% in the adrenal size of the Booroola ewes, might not be sufficient to cause a difference in the cortisol secretion profile, or, more likely, since cortisol production is so Further characterization of the development of the adrenal gland in the sheep is required. However, these results indicate that BMP signalling could be important in adrenal development, and point to the importance this family of proteins for studies of stem cell derivation particularly for adrenal lineage. Acknowledgements This work was supported by JRF Ltd Research Fellowship /3 and MRC program grant G We thank Dr Margot NA Dode for reading the manuscript and for the helpful comments. We are grateful to Ms Brigid Orr and Ms Caroline Mutch for technical assistance and to Ms Joan Docherty, Ms Norah Anderson and Ms Marjorie Thomson for care of the animals. References Baird DT, Campbell BK 1998 Follicle selection in sheep with breed differences in ovulation rate. 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5 Souza CJ, Campbell BK, Baird DT 1996 Follicular dynamics and ovarian steroid secretion in sheep during anoestrus. Journal of Reproduction and Fertility 108, Souza CJ, MacDougall C, Campbell BK et al The Booroola (FecB) phenotype is associated with a mutation in the bone morphogenetic receptor type 1 B (BMPR1B) gene. Journal of Endocrinology 169, R1 6. Souza CJ, Campbell BK, McNeilly AS et al Bone morphogenetic proteins and folliculogenesis: lessons from the Booroola mutation. Reproduction Supplement 61, Upadhyay S, Zamboni L 1982 Preliminary observations on the role of the mesonephros in the development of the adrenal cortex. Anatomical Record 202, Wilson T, Wu XY, Juengel JL et al Highly prolific Booroola sheep have a mutation in the intracellular kinase Domain of bone morphogenetic protein IB receptor (ALK-6) that is expressed in both oocytes and granulosa cells. Biology of Reproduction 64, Yi SE, Daluiski A, Pederson R et al The type I BMP receptor BMPRIB is required for chondrogenesis in the mouse limb. Development 127, Yi SE, LaPolt PS, Yoon BS et al The type I BMP receptor BmprIB is essential for female reproductive function. Proceedings of the National Academy of Sciences of the USA 98, Received 3 July 2003; refereed 7 August 2003; accepted 15 January