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BOR - Papers in Press, published online ahead of print August 22, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.062752
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BIOLOGY OF REPRODUCTION 77, 990–998 (2007)
DOI: 10.1095/biolreprod.107.062752
© 2007 by the Society for the Study of Reproduction, Inc.

Patterns of Expression of Messenger RNAs Encoding GDF9, BMP15, TGFBR1, BMPR1B, and BMPR2 During Follicular Development and Characterization of Ovarian Follicular Populations in Ewes Carrying the Woodlands FecX2W Mutation1

Elisabeth S Feary 3, Jennifer L Juengel 2 3, Peter Smith 3, Michelle C French 4, Anne R O'Connell 5, Stephen B Lawrence 3, Susan M Galloway 4, George H Davis 5, and Kenneth P McNatty 3 6

Wallaceville Animal Research Centre,3 AgResearch, Upper Hutt 5140, New Zealand Molecular Biology Unit,4 AgResearch, Dunedin 9054, New Zealand Invermay Agricultural Centre,5 AgResearch, Mosgiel 9053, New Zealand School of Biological Sciences,6 Victoria University of Wellington, Wellington 6140, New Zealand

ABSTRACT

Woodlands sheep have a putative genetic mutation (FecX2W) that increases ovulation rate. At present, the identity of FecX2W is unknown. The trait does not appear to be due to the previously described mutations in bone morphogenetic protein 15 (BMP15), growth differentiation factor 9 (GDF9), or bone morphogenetic protein receptor type 1B (BMPR1B) that affect ovulation rate in sheep. Potentially, FecX2W could be an unidentified genetic mutation in BMP15 or in the closely related GDF9, which interacts with BMP15 to control ovarian function. Alternatively, FecX2W may affect ovulation rate by changing the expression patterns in the molecular pathways activated by genes known to regulate ovulation rate. The objectives of these experiments were to sequence the complete coding region of the BMP15 and GDF9 genes, determine the patterns of expression of mRNAs encoding GDF9, BMP15, TGFBR1, BMPR1B, and BMPR2 during follicular development, and characterize the follicular populations in ewes heterozygous for the Woodlands mutation and their wild-type contemporaries. No differences in the coding sequences of BMP15 or GDF9 genes were identified that were associated with enhanced ovulation rate. The expression patterns of GDF9 and BMPR2 mRNAs were not different between genotypes. However, expression of BMP15 mRNA was less in oocytes of FecX2W ewes in large preantral and antral follicles. Expression of ALK5 mRNA was significantly higher in the oocytes of FecX2W ewes, whereas expression of BMPR1B was decreased in both oocytes and granulosa cells of FecX2W ewes. FecX2W ewes also had increased numbers of antral follicles <1 mm in diameter. These follicles were smaller in average diameter, with the oocytes also being of a smaller mean diameter. Given that a mutation in BMP15 or BMPR1B results in increased ovulation rates in sheep, the differences in expression levels of BMP15 and BMPR1B may play a role in the increase in ovulation rate observed in Woodlands ewes with the FecX2W mutation.

follicle, gene regulation, ovary, transforming growth factor beta superfamily

INTRODUCTION

Several lines of sheep have been identified with naturally occurring genetic mutations affecting ovulation rate [1]. We along with others have identified three separate but functionally related genes as being associated with regulation of follicular growth and ovulation rate [2–7]. In Inverdale, Hanna, Cambridge, and Belclare sheep, several point mutations have been identified in the bone morphogenetic protein 15 (BMP15, also known as GDF9B) gene [2, 7], and in some Cambridge ewes, a single point mutation has been identified in the closely related gene, growth differentiation factor 9 (GDF9) [7]. Each of the above point mutations results in an increased ovulation rate in heterozygous carriers, whereas primary ovarian failure is observed in those that are homozygous carriers. In Booroola, Garole, Hu, Han, and Javanese sheep, a common point mutation in the bone morphogenetic protein receptor type 1B (BMPR1B, also known as ALK6) gene has been identified [6, 8], and an increased ovulation rate/litter size is observed in ewes that are heterozygous for the mutation, with further increases observed in ewes that are homozygous carriers. BMPR1B has been identified as a potential receptor for BMP15 [9], suggesting that the underlying mechanisms used to increase ovulation rate in these lines of sheep may be very similar. In support of this hypothesis, the effects of inheriting both a BMP15 and BMPR1B mutation results in a greater-than-additive effect on ovulation rate [10]. In addition, many of the functional changes observed in follicles of ewes with mutations in BMPR1B, such as increased responsiveness to FSH and an earlier maturation of the granulosa cells, as indicated by an earlier onset of LH-R expression, are also observed in follicles from ewes with mutations in the BMP15 gene [11]. In addition, effects on oocyte development are observed in ewes with mutations in the BMP15 [10] or BMPR1B gene [4, 12], as well as in ewes actively immunized against BMP15 or GDF9 [13]. Thus, these unique sheep lines have highlighted the role of members of the transforming growth factor-β superfamily in regulation of follicular growth and ovulation rate.

Recently, we have identified two additional lines of sheep with inheritance patterns consistent with a major mutation in a gene(s) leading to an increase in ovulation rate by 0.2–0.4, namely, the Woodlands and the Metherell lines [14, 15]. Both of these lines were established from the same founding bloodlines of Coopworth sheep in the 1960s, and thus share a common ancestry, although they have been independently selected over the past 6–10 generations. These lines of sheep are extremely unusual in that the inheritance pattern of ovulation rate suggests that the mutation is located on the X chromosome and is maternally imprinted [14, 15]. This imprinting extends not only to silencing of the gene in daughters inheriting the putative gene (termed Woodlands gene hereafter) from their dams, but also to silencing in daughters of rams that have inherited it from a dam actively expressing the Woodlands gene. Therefore, increased ovulation rate only occurs in ewes that have inherited the Woodlands gene from a sire who inherited it from a dam that was a carrier but did not have an increased ovulation rate (i.e., a "silenced" carrier). Based on this very unusual inheritance pattern, it is most likely that the gene responsible in both of these lines of sheep (i.e., Woodlands and Metherell) is identical. However, whether the mutation underlying the observed phenotype is different between Woodlands and Metherell lines, as has been found with the BMP15 mutations in Inverdale, Hanna, Belclare, and Cambridge sheep [2, 7], or identical, as has been found in the BMPR1B mutations in Booroola, Garole, Hu, Han, and Javanese sheep [6, 8], remains to be determined.

The mechanisms by which the Woodlands gene is affecting ovulation rate are unknown. To our knowledge, the complex pattern of inheritance described above has not previously been described for any known mutation affecting ovulation rate in any species. The carrier animals have been tested for the mutations previously found in the Booroola and Inverdale lines [7], and these mutations are not present. However, given that the Woodlands mutation is found on the X-chromosome, it is possible that other as-yet unidentified mutations in BMP15 may underlie the observed increase in ovulation rate in these ewes. As GDF9 and BMP15 function cooperatively to regulate granulosa cell function [16, 17] and ovulation rate [7], the potential exists for the ovulation rate increases in Woodlands ewes to arise from hitherto unidentified mutations in either GDF9 or BMP15. Alternatively, the mutation may affect ovulation rate by changing the expression patterns of the BMPR1B, BMP15, or GDF9 genes. In addition, since BMPR2 is involved in signal transduction for both BMP15 [18] and GDF9 [9], and TGFBR1 is involved in signal transduction for GDF9 [19], changes in expression pattern of these genes might also affect ovulation rate. Therefore, the objectives in these experiments were to determine the full coding sequence of BMP15 and GDF9 genes in Woodlands animals and examine the patterns of expression of mRNA encoding GDF9, BMP15, TGFBR1, BMPR1B, and BMPR2 during follicular development in ewes heterozygous for the Woodlands mutation and their wild-type contemporaries. Given that different ovarian follicular characteristics are evident in sheep with mutations in BMP15 and BMPR1B [11], the follicular populations in Woodlands expresser carriers and wild-type contemporaries in the present study were examined using standard morphometric methodologies.

MATERIALS AND METHODS

All experiments were performed in accordance with the 1999 Animal Welfare Act Regulations of New Zealand. All animals had ad libitum access to pasture and water. Except where indicated, laboratory chemicals were obtained from BDH Chemicals New Zealand Ltd. (Palmerston North, New Zealand), Invitrogen (Auckland, New Zealand), or Roche Diagnostics N.Z. Ltd. (Auckland, New Zealand).

Identification of Carrier Woodlands Ewes

Matings between Coopworth ewes and fully phenotyped [15] Woodlands rams (two W/Y expresser, six +/Y rams) were used to generate Woodlands expresser (W+) ewes and wild-type contemporaries (++). As the Woodlands gene is located on the X chromosome, all daughters of an expresser Woodlands ram carry the gene in the active form. Two of the Coopworth ewes used to generate the W+ ewes were known to be from maternal sheep lines with the Woodlands gene and thus could have been carriers of the Woodlands gene. However, as the Woodlands gene is maternally imprinted, any Woodlands gene inherited from the maternal side has been silenced. Between January and March (summer to early autumn), ovaries were collected from age-matched ewes (n = 6 per genotype) between 8 and 9 years of age. Although these ewes were older than most in commercial flocks, our flock records revealed that ovulation rates of 8- to 9-year-old ewes were 5% higher than those of 2- to 7-year-old ewes, and litter sizes were identical. One ovary from each ewe was fixed in paraformaldehyde for the in situ hybridization studies, whereas the other ovary was fixed in Bouin fixative for morphometric analyses.

Sequencing of the BMP15 and GDF9 Genes in Carrier and Noncarrier Animals

DNA was isolated from the venous blood samples of four Woodlands carrier animals (W+) and two contemporary noncarrier animals (++) from the Woodlands line. DNA encoding fragments of GDF9 and BMP15 were generated using primers B-13, B-28, B-25, B-4, G-1, G-4, G-5, G-7, and G-9 as described [7]. Additional sequence was obtained using primers designed from published sheep sequences (sheep genomic BMP15 exon 1, AF236078; sheep genomic BMP15 exon 2, AF236079; sheep genomic GDF9 exons 1 and 2, AF078545; B-41, 5'-CTCAGAGTGTTCAGAAGACC-3'; B-20, 5'-CATGATTGGGAGAATTGAGACC-3'; G-9, 5'-GAAGCCTTCACAGGGTCCTGAC-3'; and G-10, 5'-TGACTGAAGCTGGAACCAGAGG-3'. The resulting PCR products were sequenced on an ABI3100 genetic analyzer (Applied Biosystems, Foster City, CA).

In Situ Hybridization

Cellular localization of mRNAs was determined using the in situ hybridization protocols described previously [2, 4, 20, 21]. The ovine GDF9 cDNA was generated from ovarian total cellular RNA by RT-PCR using the primers 5'-CCTCGAGAAAAGAGAGGCTGAAGCTGACCAGGAGAGTGCCAGCTCTGAA-3' and 5'-GGGAATTCTTAATGGTGATGGTGATGGTGACGACAGGTACACTTAGTGGCTATCA-3' (corresponds to bases 3859 to 3882 and 4238 to 4263 of the ovine genomic sequence [22] plus additional bases for subcloning) with the following conditions: 35 cycles of denaturation at 94°C, 30 sec, annealing at 55°C, 1 min, extension at 72°C for 2 min, with a final extension of 10 min at 72°C. The resulting product was ligated into pGEMTeasy and sequenced to confirm identity. Sense and antisense RNA probes were generated from cDNA encoding the gene of interest with T7 or SP6 RNA polymerase using the Riboprobe Gemini system (Promega).

For all in situ hybridizations, 4- to 6-µm tissue sections were incubated overnight at 50–55°C with 45 000 cpm/µl (approximately 48 000 dpm/µl) of 33P-labeled antisense RNA. Nonspecific hybridization of RNA was removed by RNase A digestion, followed by stringent washes (2x SSC, 50% formamide, 65°C; and 0.2x SSC at 37°C). After washing, sections were dehydrated, air dried, and coated with autoradiographic emulsion (LM-1 emulsion; Amersham Pharmacia Biotech New Zealand). Emulsion-coated slides were exposed at 4°C for 2–3 wk and developed for 3.5 min in D19 developer (Eastman Kodak, Rochester, NY). Development was stopped using a 1-min incubation in 1% acetic acid, and slides were fixed with a 10-min incubation in Ilfofix II (Ilford Limited, Cheshire, England). Sections were stained with hematoxylin and then viewed and photographed using both light and dark field illumination on an Olympus BX-50 microscope (Olympus New Zealand Ltd., Lower Hutt, New Zealand). Specific hybridization was determined for each gene in all follicular types (types 1–5) [23] in at least three animals for each genotype. Nonspecific hybridization was monitored by hybridizing at least two tissue sections from each genotype with approximately equal concentrations of the sense RNA for each gene. For all genes, hybridization of the sense RNA over the tissue section was similar or lower in intensity to that observed on the areas of the slide not containing tissue of both the sense and antisense hybridized slides, and thus was considered nonspecific.

Quantification of Level of Gene Expression

Intensity of the hybridization signal was determined by counting the number of silver grains in the cell(s) of interest using the touch count function of the Analysis software (version 3.2; Olympus Soft Imaging Solutions, Lower Hutt, New Zealand) following capture of the image with an Olympus DP12 camera. As GDF9 and BMP15 are expressed exclusively in the oocyte, signal for these genes was only quantified in this cell within the follicle. For TGFBR1, BMPR1B, and BMPR2, counts were made separately in the oocyte, granulosa, and theca cells. Measurements in theca were only undertaken for types 3–5 follicles due to the difficulty in discerning theca in types 1 and 2 follicles. The area for counting was chosen at random. The area of the cell(s) of interest was calculated using the Analysis software and the intensity of silver grains expressed as a function of this area. Between one and three slides were analyzed per animal. Moreover, each follicular type (i.e., types 1–5) was observed in at least three animals for each genotype. On each slide, up to five follicles of each type were analyzed. If more than five follicles of one type were present, five follicles were chosen at random for analysis. If five or fewer follicles of a particular type were present on a slide, all follicles of that type were analyzed. Background hybridization was determined for each slide by choosing five areas that did not contain tissue and determining the silver grain intensity in these areas. When available, these areas were in the antrum of type 5 follicles; otherwise, areas of the glass slide just outside of the ovarian cortex were chosen.

Morphometric Analyses

Ovaries were serially sectioned (5 µm), and every 10th and 11th section was mounted on a slide and stained with hematoxylin and eosin. Areas of the whole ovary as well as the ovarian cortex were estimated using point counting with a 10-mm grid for the cortex and a 20-mm grid for the whole ovary, with every 10th slide being measured (with the first slide examined being a random slide between 1 and 10). The ovarian cortex was defined as the outer region of the ovary containing all the germ cells. Volume (V) was calculated in µm3 by the Cavalieri principle [24, 25] using the formula V = {sum} a·h, where a = the cross-sectional area and h = the distance between sections (µm).

The numbers of type 1/1a (primordial) follicles were estimated using the dissector method [26–28]. A minimum of 10 slides were examined per ovary. An area of cortex was chosen randomly and measured (a(f)). Type 1/1a follicles (using the oocyte nucleus as a reference) were identified in the first section and those in the adjacent section that were not identified in the first section were counted (Q). The total follicular count (n) for each follicular type was derived using the formula n = [{sum}Q/{sum}(a(f)h(p))]V, where h(p) = the distance between each pair. For types 2, 3, 4, and 5 follicles, every slide was examined and all follicles were counted. Given the known diameter of follicles and the distance between sections mounted on slides, this method will only detect a proportion of the type 2 follicles, whereas the larger types 3, 4, and 5 follicles should be all detected. All follicles that were counted were classified as nonatretic or atretic. An atretic classification was given if the follicle contained any of the following: >10 pyknotic theca cells, >5 pyknotic granulosa cells, a disorganized thecal or granulosa layer, loss of basement membrane integrity, or a deformed oocyte nucleus.

The diameters of follicles and oocytes were measured using a Leica DMR microscope (Global Science, Auckland, New Zealand) linked to Analysis software (version 3.2). For each follicle, two measurements of diameter were made at right angles to each other and then averaged.

Statistical Analyses

Numbers of follicles were analyzed using a generalized linear model with a Poisson distribution and a logarithmic link to model the data. Oocyte and follicular diameters were transformed (natural log) before applying ANOVA, and ewe was included as a block. Differences in the proportion of atretic type 5 follicles were analyzed using a generalized linear model with a binomial distribution and a logit link to model the data. Concentrations of mRNA-encoding genes of interest were analyzed with two-way ANOVA. In all cases, a P value of < 0.05 was considered significant.

RESULTS

DNA Sequences of BMP15 and GDF9 in W+ and ++ Ewes

Sequencing of the coding DNA for BMP15 in heterozygous W+ carries and wild-type ++ sheep revealed no single-nucleotide polymorphisms (SNPs) or insertions or deletions. Sequencing of the coding DNA for GDF9 in heterozygous W+ carriers and wild-type ++ sheep revealed one SNP, which has been previously described as variant G5 [7] in one W+ and one ++ animal. G5 is an A–G nucleotide substitution at coding base position 978 that leaves the glutamate residue unchanged and which has been seen in a number of sheep from a variety of breeds. No insertions or deletions were identified in GDF9. None of the previous functional mutations observed in these genes in Inverdale, Hanna, Belclare, or Cambridge sheep were present.

Expression of mRNAs in W+ and ++ Ewes

GDF9 and BMP15. Concentrations of GDF9 mRNA in oocytes were different (P ≤ 0.01) in different types of follicles, with an approximately 2-fold increased expression observed between types 1 and 2 follicles, but no differences were found between genotypes (Fig. 1). The concentrations of BMP15 mRNA in oocytes differed (P < 0.001) among types of follicles (Fig. 1), with expression increasing approximately 2-fold as follicles progressed from type 2 to 4 in maturation. There was a trend toward a genotype effect (P = 0.102) on BMP15 mRNA expression, but no interaction between genotype and follicular type. However, a genotype difference was noted when the data for type 4 and 5 follicles were combined (P < 0.05), with a 30% decrease in BMP15 mRNA expression in W+ compared with ++ oocytes (Fig. 2).


Figure 01
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  		FIG. 1 Mean (±SEM) density of silver grains following hybridization with RNA to detect mRNA encoding GDF9 (top) and BMP15 (bottom) in oocytes of types 1, 2, 3, 4, and 5 follicles from ewes that were heterozygous carriers of an active Woodlands gene (W+; solid bars) and their wild-type contemporaries (++; open bars). BMP15 mRNA was below the level of detection in type 1 follicles.


Figure 02
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  		FIG. 2 Corresponding brightfield (A, C, E, G) and darkfield (B, D, F, H) views of ovaries from control (++) ewes (A, B, E, F) and ewes expressing an active copy of the Woodlands gene (W+; C, D, G, H) following hybridization to BMP15 antisense RNA (A–D) or BMPR1B (E–H) antisense RNA. Note the decreased signal for BMP15 mRNA in the oocyte of the early type 5 follicles in the ovary of a W+ ewes (C, D) compared with expression in the oocyte of a late type 4 follicle in the ovary of a ++ ewe (A, B). Similarly, expression of BMPR1B mRNA is less in the granulosa cells of an antral follicle from a W+ ewe (G, H) compared with a ++ ewe (E, F).

TGFBR1 mRNA. Both genotype (P < 0.05) and follicular type (P < 0.01) affected the concentrations of TGFBR1 mRNA in the oocyte, but no interactions between genotype and follicular type were noted (Fig. 3). Concentrations of TGFBR1 mRNA in oocytes were higher in types 1–4 follicles (0.018–0.28 silver grains/µm2) than in type 5 follicles (0.006 silver grains/µm2). Overall, the concentration of TGFBR1 mRNA expressed in oocytes of ++ follicles was 19% less than expression in oocytes of W+ follicles. In granulosa cells, the concentrations of TGFBR1 differed among follicular types (P < 0.01) but were not affected by genotype (Fig. 3). Expression of TGFBR1 mRNA in granulosa cells doubled between types 1–3 follicles, and intensity of expression appeared to reach a plateau at this stage. Concentrations of TGFBR1 mRNA were consistently low in the theca, with no effects of either genotype or follicular type noted (Fig. 3).


Figure 03
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  		FIG. 3 Mean (±SEM) density of silver grains following hybridization with RNA to detect mRNA encoding TGFBR1 in oocytes (top), granulosa cells (middle), and theca (bottom) of types 1, 2, 3, 4, and 5 follicles from ewes that were heterozygous carriers of an active Woodlands gene (W+; solid bars) and their wild-type contemporaries (++; open bars).

BMPR1B mRNA. Expression of BMPR1B mRNA in the oocyte was affected by both follicular type (P < 0.001) and genotype (P < 0.01), but there were no interactions (Fig. 4). Concentrations of BMPR1B mRNA in the oocyte peaked in type 2 follicles, with expression being lower in types 4 (0.006 silver grains/µm2) and 5 (0.003 silver grains/µm2) follicles compared with types 1 (0.017 silver grains/µm2), 2 (0.022 silver grains/µm2), and 3 (0.015 silver grains/µm2) follicles. Overall, the expression of BMPR1B mRNA was 24% greater in ++ compared with W+ oocytes, with this difference being most evident in the type 1 follicles. Concentrations of BMPR1B mRNA in granulosa cells were also affected by both genotype (P < 0.01) and follicular type (P < 0.01), but no interactions were observed (Fig. 4). Expression was very low in type 1 follicles (0.007 silver grains/µm2), with an increased level of expression observed in type 2 follicles (0.025 silver grains/µm2). The concentration of BMPR1B mRNA in granulosa cells of type 2 follicles was slightly increased compared with types 3 (0.017 silver grains/µm2) and 4 (0.019 silver grains/µm2) but not type 5 (0.023 silver grains/µm2) follicles. Overall, a small (21%) but consistent decreased expression of BMPR1B mRNA in the granulosa cells of W+ follicles was observed compared with granulosa cells of ++ follicles (Fig. 2). Low concentrations of BMPR1B mRNA were consistently expressed in the theca, with no effect of either genotype or follicular type observed (Fig. 4).


Figure 04
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  		FIG. 4 Mean (±SEM) density of silver grains following hybridization with RNA to detect mRNA encoding BMPR1B in oocytes (top), granulosa cells (middle), and theca (bottom) of types 1, 2, 3, 4, and 5 follicles from ewes that were heterozygous carriers of an active Woodlands gene (W+; solid bars) and their wild-type contemporaries (++; open bars).

BMPR2 mRNA. Neither genotype nor follicular type affected concentrations of BMPR2 mRNA in the oocytes (Fig. 5). In granulosa cells, concentrations of BMPR2 mRNA were affected by follicular type (P < 0.01) but not genotype (Fig. 5). Concentrations of BMPR2 mRNA in granulosa cells increased slightly between types 1 (0.020 silver grains/µm2) and 2 (0.027 silver grains/µm2) follicles, fell significantly in types 3 (0.018 silver grains/µm2) and 4 (0.019 silver grains/µm2) follicles, but then increased such that expression in type 5 follicles (0.020 silver grains/µm2) was not different from that observed at the 1, 3, or 4 stages, although concentrations did not reach the peak observed in type 2 follicles. Expression of BMPR2 mRNA in theca differed with respect to follicular type (P < 0.05) but not genotype (Fig. 5). Expression of BMPR2 mRNA was greater in type 3 than in type 4 follicles, with expression in type 5 follicles being intermediate.


Figure 05
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  		FIG. 5 Mean (±SEM) density of silver grains following hybridization with RNA to detect mRNA encoding BMPR2 in oocytes (top), granulosa cells (middle), and theca (bottom) of types 1, 2, 3, 4, and 5 follicles from ewes that were heterozygous carriers of an active Woodlands gene (W+; solid bars) and their wild-type contemporaries (++; open bars).

Characterization of Ovarian Follicles in W+ and ++ Ewes

Numbers and measurements of follicles. The numbers of nonatretic types 1–4 follicles did not differ between genotypes. However, W+ ewes had an increased number (P < 0.05) of nonatretic type 5 (antral) follicles compared with ++ ewes (Table 1). Further characterization of the type 5 follicles indicated that the W+ ewes had approximately twice as many (P < 0.05) type 5 follicles ≤1 mm in diameter compared with ++ ewes. However, there were no differences between the genotypes in the number of follicles >1 mm in diameter. Genotype did not affect the number of atretic follicles, but due to the increased number of nonatretic type 5 follicles in W+ ewes, the proportion of type 5 follicles that were atretic was lower (P < 0.01) in W+ ewes than in ++ ewes. The mean diameters of types 1–4 follicles or oocytes did not differ between genotypes (Table 1). However, the mean diameter of type 5 follicles in W+ ewes was smaller compared with that in ++ ewes (P < 0.001). The mean diameter of the oocyte also was smaller in type 5 follicles of W+ ewes compared with ++ ewes (P < 0.001). However, when oocyte diameter was expressed as a fraction of follicular diameter, the oocytes in type 5 follicles were proportionally larger (P < 0.01) in W+ ewes than in ++ ewes.


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  		TABLE 1 Geometric mean and 95% confidence limits for the number of follicles, oocyte diameter, and follicular diameter for each follicular type in both wild-type (++) and heterozygous Woodlands (W+) ewes.a

Follicular morphology. Differences in morphology of type 5 follicles were noted between genotypes (Fig. 6). Many (36%) of the type 5 follicles ≤1 mm in diameter in the W+ ewes were unusual in their shape. For example, the antrum did not display the usual pattern of development and the number of layers of granulosa cells was more consistent with that normally observed in type 4 follicles. Fewer than 1% of the follicles ≤1 mm in diameter in the ++ ewes had this unusual morphology. These unusual follicles were not atretic using the above described criteria for atresia.


Figure 06
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  		FIG. 6 Photo micrographs of ovaries from wild-type (++) and Woodlands carrier (W+) ewes. A, B) Nonatretic follicle 0.43 mm in diameter from a ++ ovary showing normal theca (t), healthy granulosa cell layer (g), and typical centrally located developing antrum (a) separated from the basement membrane by multiple cell layers. C–F) Nonatretic follicles collected from two W+ ewes. The follicle shown in C and D is 0.27 mm in diameter, whereas that in E and F is 0.38 mm in diameter. In C and E, arrows indicate disorganized theca area adjacent to developing antrum. In D and F, note also unusual development of the antrum further illustrated by arrows.

DISCUSSION

Genetic differences in the ovulation rate of Woodlands ewes have previously been reported [15], but the physiologic mechanisms that underlie the increase in ovulation rate in FecX2W ewes are not known. As the inheritance pattern of ovulation rate is consistent with a mutation located on the X chromosome, it is possible that an unidentified mutation in BMP15, a gene known to regulate ovulation rate in sheep, could be responsible for the increased ovulation rate. However, complete sequencing of the coding sequence of the BMP15 gene failed to link any variation in the sequence of this gene to the increased ovulation rate observed, indicating that a mutation in this region of the BMP15 gene can be discounted as contributing to the enhanced ovulation rate observed in the ewes with an active copy of the Woodlands gene. Potentially, mutations in other regions of the BMP15 gene could be present.

Expression of BMP15 mRNA was decreased approximately 30% in oocytes of W+ ewes compared with expression in ++ ewes. Although the differences in BMP15 mRNA expression were only seen in late preantral and in antral follicles, BMP15 is known to be essential for the later as well as the early stages of follicular development in sheep [13]. Multiple mutations in BMP15, with potentially differing actions on the biologic activity of the resulting protein, have been identified. Based on the differing effects of these mutations on ovulation rate, a predictive equation for ovulation rate based on the biologic activity of BMP15 compared with a wild-type sheep has been developed [29]. Assuming that a 30% decrease in BMP15 mRNA would result in a 30% decrease in BMP15 biologic activity, the predicted ovulation rate of FecX2W ewes would be approximately 120% of wild-type ewes, which is exactly the actual increase observed in a previous study of the Woodlands line [15]. Thus, decreased concentrations of BMP15 mRNA may underlie the increased ovulation rate observed in FecX2W ewes.

Another gene that regulates ovulation rate in sheep is GDF9. Although this gene is not located on the X-chromosome, and thus is an unlikely candidate, previous studies in lines of sheep with high ovulation rates revealed multiple mutations in BMP15 and GDF9 segregating together [7]. Such co-segregation masked the ready identity of the X-linked component in segregation studies. Therefore, given the unusual pattern of inheritance seen in Woodlands sheep, it was important to rule out the possibility that alterations in the sequence and function of this gene might affect ovulation rate, either directly or indirectly. However, no sequence variations linked to ovulation rate were found in the coding region of GDF9. Moreover, no genotype differences were observed in the concentrations of GDF9 mRNA expressed in oocytes, indicating that there are no overall net differences resulting from the effects of transcription and degradation of the mRNA.

It is possible that changes in expression of genes for the receptors of either GDF9 or BMP15, namely, BMPR2, TGFBR1, and BMPR1B, could influence ovulation rate in ewes expressing the Woodlands gene. However, as with GDF9, none of these genes are located on the X-chromosome, so their effects are likely to be indirect. While no differences were observed in the expression of BMPR2 mRNA between W+ and ++ ewes, differences were observed in the expression of TGFBR1 in the oocyte and BMPR1B in both the oocyte and granulosa cells.

The mechanisms by which alterations in expression of mRNA for the type I receptors for GDF9 (i.e., TGFBR1) and BMP15 (i.e., BMPR1B) in oocytes might affect ovulation rate is unknown. GDF9 has been implicated in the transition of a follicle from a preantral to an antral stage, with the in vitro addition of oocytes or GDF9 inducing many of the changes observed in granulosa cells progressing from preantral to antral stages of development [30]. This appears to be an important transitional stage in follicular development. In mice, this stage signifies the differentiation of granulosa cells into cumulus and mural granulosa cell phenotypes, each with more specialized functions. The increased expression of TGFBR1 mRNA observed in the oocytes of the ewes expressing the Woodlands gene could be associated with advancing the development of the cumulus and mural granulosa cell phenotypes. In the present study, significantly more follicles in the W+ ewes showed evidence of earlier formation of an antrum with fewer layers of granulosa cells. This may be a consequence of enhanced signaling by GDF9 due to higher concentrations of TGFBR1 mRNA in the oocyte.

Expression of BMPR1B mRNA was also altered during follicular development. A mutation in BMPR1B that is known to increase ovulation rates in ewes heterozygous or homozygous for the mutation has previously been identified [29]. The mutated receptor has been shown to be less responsive to BMPs, suggesting that the mutation decreases the signaling ability of the receptor although basal activity of the receptor may be increased slightly [31]. A decreased sensitivity of granulosa cells to BMP15 due to decreased expression of BMPR1B might provide a similar physiologic signal, as observed in ewes heterozygous for inactivating BMP15 mutations or heterozygous or homozygous for the identified BMPR1B mutation. Further support that the decreased level of BMPR1B mRNA expression might underlie, at least in part, the increased ovulation rate observed in these ewes comes from the known interaction of these genes in regulating ovulation rate in sheep in vivo [32]. Ewes that carry mutations in both the BMPR1B and BMP15 genes, as well as the Woodlands allele, have very high ovulation rates, and the effects of the genes appear synergistic [32]. Thus, it appears that all three genes alter ovulation through the same or interacting pathways.

The numbers of preantral follicles were not different between W+ and ++ ewes. As BMP15 and GDF9 are essential for early follicular development, preantral follicular development is impaired in ewes homozygous for mutations in either gene [7, 33, 34]; however, no differences were observed in the numbers of primordial or primary follicles between heterozygous (I+) Inverdale ewes compared with wild-type controls [27], indicating that these growth factors do not regulate the numbers of follicles formed or entering the growing pool. In contrast, the numbers of preantral follicles have been shown to be increased in ewes homozygous (BB) for the Booroola mutation [35].

Ewes expressing the Woodlands gene had increased number of small antral follicles. Differences in the numbers of antral follicles <1 mm in diameter has not been examined in I+ ewes; however, increased numbers of follicles 1–2.5 mm, but not >2.5 mm, in diameter have been observed in I+ animals [36]. Moreover, as the proportion of nonatretic follicles in I+ ewes was not different from the ++ ewes, I+ animals appear to have more nonatretic follicles available to be recruited for ovulation. This is consistent with the higher ovulation rates observed after superovulation treatments in I+ compared with ++ ewes [37]. However, ewes immunized with BMP15 that continued to have reproductive cycles had numbers of antral follicles <1 mm in diameter similar to those in control immunized ewes [13]. Similarly, the number of antral follicles do not differ between BB and ++ ewes [35]. Thus, while alterations in numbers of follicles at specific developmental stages are observed in ewes carrying the Woodlands, Inverdale, or Booroola gene, the developmental stages at which these differences are observed were different among the three lines of sheep.

Overall, the diameters of oocytes and follicles at the type 5 stage were smaller in W+ follicles than ++ follicles. The smaller diameter of type 5 follicles observed in W+ ewes was most likely related to the increased numbers of type 5 follicles <1 mm in diameter. When oocyte diameter was examined as a proportion of follicle size, oocytes in W+ ewes were actually larger than those observed in wild-type contemporaries. An increase in oocyte diameter for follicle size is a common feature of both Booroola and Inverdale ewes as well as ewes immunized against GDF9 or BMP15 [4, 12, 13, 27]. It is thought that this increased oocyte size is related to an enhanced developmental stage of the ovarian follicle that is accompanied by an enhanced responsiveness of the follicles to gonadotrophins.

In summary, follicular development is altered in ewes expressing the Woodlands gene. Formation of the follicular antrum occurred earlier in W+ ewes, and in many of these the follicular structures were atypical. Expression of mRNAs encoding BMP15, TGFBR1, and BMPR1B was altered in W+ ewes, potentially leading to alterations in the signaling pathways for the oocyte-derived growth factors GDF9 and BMP15. Given that mutations in GDF9, BMP15, or BMPR1B alter ovulation rates in sheep and that the Woodlands gene is known to interact with the BMP15 and BMPR1B gene to induce very high ovulation rates, it is proposed that alterations in expression of the BMP15, TGFBR1, and/or BMPR1B genes contribute to the increased ovulation rate observed in W+ ewes.

ACKNOWLEDGMENTS

The authors would like to thank Lilian Morrison and Peter Johnstone for help with statistical analyses. The assistance of Doug Jensen and Phil Farquhar with animal care and tissue collection is greatly appreciated.

FOOTNOTES

1Supported by the Marsden Fund, administered by the Royal Society of New Zealand and the Foundation for Research Science and Technology, New Zealand. E.S.F. was supported by an Enterprise Scholarship. Back

Correspondence: 2Jenny Juengel, AgResearch, Wallaceville Animal Research Centre, Ward St., Upper Hutt 5140, New Zealand. FAX: 64 4 529 0380; e-mail: jenny.juengel{at}agresearch.co.nz

Received: 9 May 2007.

First decision: 4 June 2007.

Accepted: 17 August 2007.

REFERENCES

  1. Davis GH. Major genes affecting ovulation rate in sheep Genet Sel Evol 2005 37(suppl 1)S11–S23[CrossRef][Medline]
  2. Galloway SM, McNatty KP, Cambridge LM, Laitinen MP, Juengel JL, Jokiranta TS, McLaren RJ, Luiro K, Dodds KG, Montgomery GW, Beattie AE, Davis GH, et al. Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner Nat Genet 2000 25279–283[CrossRef][Medline]
  3. Mulsant P, Lecerf F, Fabre S, Schibler L, Monget P, Lanneluc I, Pisselet C, Riquet J, Monniaux D, Callebaut I, Cribiu E, Thimonier J, et al. Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Merino ewes Proc Natl Acad Sci U S A 2001 985104–5109[Abstract/Free Full Text]
  4. Wilson T, Wu XY, Juengel JL, Ross IK, Lumsden JM, Lord EA, Dodds KG, Walling GA, McEwan JC, O'Connell AR, McNatty KP, Montgomery GW. 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 Biol Reprod 2001 641225–1235[Abstract/Free Full Text]
  5. Souza CJ, MacDougall C, Campbell BK, McNeilly AS, Baird DT. The Booroola (FecB) phenotype is associated with a mutation in the bone morphogenetic receptor type 1 B (BMPR1B) gene J Endocrinol 2001 169R1–R6[Abstract]
  6. Davis GH, Galloway SM, Ross IK, Gregan SM, Ward J, Nimbkar BV, Ghalsasi PM, Nimbkar C, Gray GD, Subandriyo, Inounu I, Tiesnamurti B, et al. DNA tests in prolific sheep from eight countries provide new evidence on origin of the Booroola (FecB) mutation Biol Reprod 2002 661869–1874[Abstract/Free Full Text]
  7. Hanrahan JP, Gregan SM, Mulsant P, Mullen M, Davis GH, Powell R, Galloway SM. Mutations in the genes for oocyte-derived growth factors GDF9 and BMP15 are associated with both increased ovulation rate and sterility in Cambridge and Belclare sheep (Ovis aries) Biol Reprod 2004 70900–909[Abstract/Free Full Text]
  8. Davis GH, Balakrishnan L, Ross IK, Wilson T, Galloway SM, Lumsden BM, Hanrahan JP, Mullen M, Mao XZ, Wang GL, Zhao ZS, Zeng YQ, et al. Investigation of the Booroola (FecB) and Inverdale (FecX(I)) mutations in 21 prolific breeds and strains of sheep sampled in 13 countries Anim Reprod Sci 2006 9287–96[CrossRef][Medline]
  9. Moore RK, Otsuka F, Shimasaki S. Molecular basis of bone morphogenetic protein-15 signaling in granulosa cells J Biol Chem 2003 278304–310[Abstract/Free Full Text]
  10. Davis GH, Dodds KG, Bruce GD. Combined effect of the Inverdale and Booroola prolificacy genes on ovulation rate in sheep Proc Assoc Advmt Anim Breed Genet 1999 1374–77
  11. McNatty KP, Juengel JL, Wilson T, Galloway SM, Davis GH, Hudson NL, Moeller CL, Cranfield M, Reader KL, Laitinen MP, Groome NP, Sawyer HR, et al. Oocyte-derived growth factors and ovulation rate in sheep Reprod Suppl 2003 61339–351[Medline]
  12. Cognie Y, Benoit F, Poulin N, Khatir H, Driancourt MA. Effect of follicle size and of the FecB Booroola gene on oocyte function in sheep J Reprod Fertil 1998 112379–386[Abstract/Free Full Text]
  13. Juengel JL, Hudson NL, Heath DA, Smith P, Reader KL, Lawrence SB, O'Connell AR, Laitinen MP, Cranfield M, Groome NP, Ritvos O, McNatty KP. Growth differentiation factor 9 and bone morphogenetic protein 15 are essential for ovarian follicular development in sheep Biol Reprod 2002 671777–1789[Abstract/Free Full Text]
  14. Davis GH, Dodds KG, Wheeler R, Bruce GD. Further evidence of Non-Mendelian inheritance at the FECX2 locus in a prolific sheep flock Proceedings 7th World Congress on Genetics Applied to Livestock Production 2002 30641–644
  15. Davis GH, Dodds KG, Wheeler R, Jay NP. Evidence that an imprinted gene on the X chromosome increases ovulation rate in sheep Biol Reprod 2001 64216–221[Abstract/Free Full Text]
  16. McNatty KP, Juengel JL, Reader KL, Lun S, Myllymaa S, Lawrence SB, Western A, Meerasahib MF, Mottershead DG, Groome NP, Ritvos O, Laitinen MP. Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function in ruminants Reproduction 2005 129481–487[Abstract/Free Full Text]
  17. McNatty KP, Juengel JL, Reader KL, Lun S, Myllymaa S, Lawrence SB, Western A, Meerasahib MF, Mottershead DG, Groome NP, Ritvos O, Laitinen MP. Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function Reproduction 2005 129473–480[Abstract/Free Full Text]
  18. Vitt UA, Mazerbourg S, Klein C, Hsueh AJ. Bone morphogenetic protein receptor type II is a receptor for growth differentiation factor-9 Biol Reprod 2002 67473–480[Abstract/Free Full Text]
  19. Mazerbourg S, Klein C, Roh J, Kaivo-Oja N, Mottershead DG, Korchynskyi O, Ritvos O, Hsueh AJ. Growth differentiation factor-9 signaling is mediated by the type I receptor, activin receptor-like kinase 5 Mol Endocrinol 2004 18653–665[Abstract/Free Full Text]
  20. Juengel JL, Bibby AH, Reader KL, Lun S, Quirke LD, Haydon LJ, McNatty KP. The role of transforming growth factor-beta (TGF-beta) during ovarian follicular development in sheep Reprod Biol Endocrinol 2004 278[CrossRef][Medline]
  21. Tisdall DJ, Fidler AE, Smith P, Quirke LD, Stent VC, Heath DA, McNatty KP. Stem cell factor and c-kit gene expression and protein localization in the sheep ovary during fetal development J Reprod Fertil 1999 116277–291[Abstract/Free Full Text]
  22. Bodensteiner KJ, Clay CM, Moeller CL, Sawyer HR. Molecular cloning of the ovine growth/differentiation factor-9 gene and expression of growth/differentiation factor-9 in ovine and bovine ovaries Biol Reprod 1999 60381–386[Abstract/Free Full Text]
  23. Lundy T, Smith P, O'Connell A, Hudson NL, McNatty KP. Populations of granulosa cells in small follicles of the sheep ovary J Reprod Fertil 1999 115251–262[Abstract/Free Full Text]
  24. Gundersen HJ. Stereology of arbitrary particles. A review of unbiased number and size estimators and the presentation of some new ones, in memory of William R. Thompson J Microsc 1986 1433–45[Medline]
  25. Gundersen HJ, Jensen EB. The efficiency of systematic sampling in stereology and its prediction J Microsc 1987 147229–263[Medline]
  26. Sterio DC. The unbiased estimation of number and sizes of arbitrary particles using the disector J Microsc 1984 134127–136[Medline]
  27. Smith P, O WS, Corrigan KA, Smith T, Lundy T, Davis GH, McNatty KP. Ovarian morphology and endocrine characteristics of female sheep fetuses that are heterozygous or homozygous for the Inverdale prolificacy gene (FecXI) Biol Reprod 1997 571183–1192[Abstract]
  28. Smith P, O WS, Hudson NL, Shaw L, Heath DA, Condell L, Phillips DJ, McNatty KP. Effects of the Booroola gene (FecB) on body weight, ovarian development and hormone concentrations during fetal life J Reprod Fertil 1993 9841–54[Abstract/Free Full Text]
  29. McNatty KP, Moore LG, Hudson NL, Quirke LD, Lawrence SB, Reader K, Hanrahan JP, Smith P, Groome NP, Laitinen M, Ritvos O, Juengel JL. The oocyte and its role in regulating ovulation rate: a new paradigm in reproductive biology Reproduction 2004 128379–386[Abstract/Free Full Text]
  30. Latham KE, Wigglesworth K, McMenamin M, Eppig JJ. Stage-dependent effects of oocytes and growth differentiation factor 9 on mouse granulosa cell development: advance programming and subsequent control of the transition from preantral secondary follicles to early antral tertiary follicles Biol Reprod 2004 701253–1262[Abstract/Free Full Text]
  31. Fabre S, Pierre A, Pisselet C, Mulsant P, Lecerf F, Pohl J, Monget P, Monniaux D. The Booroola mutation in sheep is associated with an alteration of the bone morphogenetic protein receptor-IB functionality J Endocrinol 2003 177435–444[Abstract]
  32. Davis GH. Fecundity genes in sheep Anim Reprod Sci 2004 82–83247–253
  33. McNatty KP, Galloway SM, Wilson T, Smith P, Hudson NL, O'Connell A, Bibby AH, Heath DA, Davis GH, Hanrahan JP, Juengel JL. Physiological effects of major genes affecting ovulation rate in sheep Genet Sel Evol 2005 37(suppl 1)S25–S38[CrossRef][Medline]
  34. Davis GH, McEwan JC, Fennessy PF, Dodds KG, McNatty KP, WS O. Infertility due to bilateral ovarian hypoplasia in sheep homozygous (FecXI FecXI) for the Inverdale prolificacy gene located on the X chromosome Biol Reprod 1992 46636–640[Abstract]
  35. Driancourt MA, Cahill LP, Bindon BM. Ovarian follicular populations and preovulatory enlargement in Booroola and control Merino ewes J Reprod Fertil 1985 7393–107[Abstract/Free Full Text]
  36. Shackell GH, Hudson NL, Heath DA, Lun S, Shaw L, Condell L, Blay LR, McNatty KP. Plasma gonadotropin concentrations and ovarian characteristics in Inverdale ewes that are heterozygous for a major gene (FecX1) on the X chromosome that influences ovulation rate Biol Reprod 1993 481150–1156[Abstract]
  37. McNatty KP, Hudson N, Smith P, Juengel JL. The effects of superovulating sheep with mutations in either the activin-like kinase (Alk6) or bone morphogenetic protein (BMP15) genes on ovulation rate and embryo production J Reprod Dev 2006 52S39–S43



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