Expression Analysis of the Prolific Candidate Genes, BMPR1B, BMP15, and GDF9 in Small Tail Han Ewes with Three Fecundity (FecB Gene) Genotypes

Simple Summary As important prolific candidate genes, BMPR1B, BMP15, and GDF9 may affect the lambing performance of sheep. Therefore, regarding the three FecB genotypes of Small Tail Han (STH) sheep (FecB BB, FecB B+, and FecB ++), this study explored the gene expression characteristics of different tissues using reverse transcription PCR (RT-PCR) and real-time quantitative PCR (qPCR). The results showed that BMPR1B, BMP15, and GDF9 expression differed between the selected tissues, with all being highly expressed in the ovaries. Further analysis indicated that there was no significant difference in BMPR1B expression among the three FecB genotypes, but both GDF9 and BMP15 had the highest expression in FecB B+. As for other non-ovarian tissues, expression also varied. This study is relevant to understanding the high prolificacy of the STH breed. Abstract The expression characteristics of the prolific candidate genes, BMPR1B, BMP15, and GDF9, in the major visceral organs and hypothalamic–pituitary–gonadal (HPG) axis tissues of three FecB genotypes (FecB BB, FecB B+, and FecB ++) were explored in STH ewes using RT-PCR and qPCR. The results were as follows, BMPR1B was expressed in all FecB BB genotype (Han BB) tissues, and GDF9 was expressed in all selected tissues, but BMP15 was specifically expressed in the ovaries. Further study of ovarian expression indicated that there was no difference in BMPR1B expression between genotypes, but the FecB B+ genotype (Han B+) had greater expression of GDF9 and BMP15 than Han BB and FecB ++ genotype (Han ++) (p < 0.05, p < 0.01). BMP15 expression was lower in the ovaries of Han BB than in Han ++ sheep, but the reverse was shown for GDF9. The gene expression in non-ovarian tissues was also different between genotypes. Therefore, we consider that the three genes have an important function in ovine follicular development and maturation. This is the first systematic analysis of the tissue expression pattern of BMPR1B, BMP15, and GDF9 genes in STH sheep of the three FecB genotypes. These results contribute to the understanding of the molecular regulatory mechanism for ovine reproduction.


Sample Collection
The tissue samples were collected 45-48 h after CIDR removal, based on preliminary test observations. During this period, the developing follicles were at their largest, but have not yet ovulated. Fourteen tissue samples from the heart, liver, spleen, lung, kidney, brain, cerebellum, hypothalamus, pituitary, ovary, oviduct, uterus, adrenal gland, and duodenum were obtained from sheep of the three FecB genotypes. The tissue samples were collected within 30 min of euthanasia. Fresh samples were frozen in liquid nitrogen in 2 mL RNase-Free tubes (Thermo Fisher Scientific, Waltham, MA, USA) immediately and stored at −80 • C in the laboratory.

Total RNA Extraction and cDNA Synthesis
Tissue RNA was extracted from the 14 tissues using a total RNA extraction kit for animal tissue (Tiangen, Beijing, China) and Trizol (Invitrogen Inc., Carlsbad, CA, USA) was used to dissolve the tissues. The quantity and quality of total RNA were monitored by 1.5% agarose gel electrophoresis and ultraviolet spectrophotometry (UV-1201, Shimadzu, Kyoto, Japan), respectively. Then, the RNAs were stored at -80 • C until use.
The first strand of cDNA was prepared following the instructions of the PrimeScript TM RT Reagent Kit (TaKaRa Bio Inc., Dalian, China). The reaction program was as follows. 37 • C for 15 min, followed by 85 • C for 5 s, with a total volume of 20 µL which contained 1 µg of total RNA, 1 µL of PrimeScript RT Enzyme Mix I, 1 µL of Oligo dT Primer, 1 µL of random 6-mers, 4 µL of 5× PrimeScript Buffer (for Real Time), and 12 µL of RNase-free water. Prior to storage at −80 • C, the cDNA quality was evaluated by housekeeping gene (β-actin) amplification, and then the reverse products were stored at −20 • C until use.

Primer Design
Primers were designed with Premier 3.0 (version 4.1.0) online software (http://primer3.ut.ee/). Primer sequences of BMPR1B, BMP15, GDF9, and β-actin were selected from GenBank (http://www. ncbi.nlm.nih.gov/) for RT-PCR and qPCR analyses. Previously published literature also provided a reference for the primer sequence of BMP15 for PCR [28]. All primers were synthesized by the Beijing Tianyi Biotechnology Co., Ltd. (Beijing, China). The housekeeping gene (β-actin) was used as an internal control to normalize the threshold cycle (Ct) values. Primers are detailed in Table 1.

Exploration of Gene Amplification Parameters
To explore the optimal amplification cycles for BMPR1B, BMP15, and GDF9 in RT-PCR, seven cycle parameters, 26, 28, 30, 32, 34, 36, and 38, were set to amplify the ovarian tissue cDNA by PCR.

RT-PCR Program and Amplification System
The reverse transcribed cDNA was used for the RT-PCR analysis. The volume of the amplification system of RT-PCR was 20 µL, including 10 µL of 2 × PCR Master Mix (Biomed, Beijing, China), 0.5 µL of 10 mmol/L primer (forward and reverse), 1.0 µL of cDNA, and 8 µL of ddH 2 O. The amplification program was as follows: initial denaturation at 95 • C for 5 min; followed by the optimal cycles (34, 34, and 36 cycles each for BMPR1B, BMP15, and GDF9) of denaturation at 95 • C for 30 s (see Figure 1); annealing for 30 s; and extension at 72 • C for 60 s, with a final extension at 72 • C for 5 min, and then the PCR product was stored at 4 • C.

Exploration of Gene Amplification Parameters
To explore the optimal amplification cycles for BMPR1B, BMP15, and GDF9 in RT-PCR, seven cycle parameters, 26, 28, 30, 32, 34, 36, and 38, were set to amplify the ovarian tissue cDNA by PCR.

RT-PCR Program and Amplification System
The reverse transcribed cDNA was used for the RT-PCR analysis. The volume of the amplification system of RT-PCR was 20 μL, including 10 μL of 2 × PCR Master Mix (Biomed, Beijing, China), 0.5 μL of 10 mmol/L primer (forward and reverse), 1.0 μL of cDNA, and 8 μL of ddH2O. The amplification program was as follows: initial denaturation at 95 °C for 5 min; followed by the optimal cycles (34, 34, and 36 cycles each for BMPR1B, BMP15, and GDF9) of denaturation at 95 °C for 30 s (see Figure 1); annealing for 30 s; and extension at 72 °C for 60 s, with a final extension at 72 °C for 5 min, and then the PCR product was stored at 4 °C.   Tissue qPCR was performed in triplicate along with the negative controls (H 2 O used as template) using the Roche Light Cycler ® 480 II. The qPCR amplification mixture was 20 µL, containing 10 µL of SYBR ® Premix ExTaq II (TaKaRa Bio Inc., Dalian, China), 0.8 µL of primers (forward and reverse), 2 µL of cDNA, and the rest being ddH 2 O. The qPCR program is as follows, with the initial denaturation at 95 • C for 5 min, followed by 40 cycles of denaturation at 95 • C for 5 s and 60 • C for 30 s. The gene expression data were normalized to the β-actin gene. After amplification, the melting curve was analyzed.

Establishment of the Standard Curve
One microliter of cDNA was pipetted from each sample. Then, eight gradient concentrations of cDNA were mixed and diluted: 1-, 2-, 4-, 8-, 16-, 32-, 64-, and 128-fold. BMPR1B, BMP15, GDF9, and β-actin were quantified by qPCR using this diluted cDNA as templates. The standard curves (Supplementary File 1) of the target and housekeeping genes were drawn as the C t value (0-40) against the cDNA concentration (natural logarithm).

Data Statistics
The relative gene expression levels were calculated by the 2 −∆∆Ct method [29,30]. Statistical analyses were carried out using SPSS 22.0 software (IBM Armonk, NY, USA). The levels of gene expression were analyzed for significant differences with one-way analysis of variance (ANOVA), followed by the Fisher's least significant difference (LSD) test as a multiple comparison test [31]. All experimental data are shown as mean ± SEM. Statistical significance was taken as p < 0.05.

RNA Extraction and cDNA Synthesis
Total RNA samples were analyzed using 1.5% agarose gel electrophoresis (U = 150 V; I = 240 mA). Three bands (representing 28S, 18S, and 5S) were detected-the 28S band was brighter than the 18S band, and the 5S band was unclear. The OD260 nm/OD280 nm ratios (1.8-2.0) of the samples RNA were all 1.9 to 2.0, which showed that the extracted total RNA was of sufficient purity with no contamination or degradation. Therefore, these tissue RNAs could be used in the follow-up experiment.

Exploration of the Amplification Parameters
In principle, to explore the optimal amplification parameters, it is important that the luminance of the gene amplification band remains unchanged with the increase in reaction cycles. As seen in Figure 1, the selective cycles of BMPR1B, BMP15, and GDF9 were 34, 36, and 36, respectively.

Tissue-Specific Expression Analysis of BMPR1B, BMP15, and GDF9
The results of the RT-PCR amplification showed that the fragment lengths of the amplification products of BMPR1B, BMP15, GDF9, and β-actin were consistent with their theoretical lengths, identified by agarose gel electrophoresis (1.5%). As seen in Figure 2, the β-actin gene, as the reference gene, was successfully expressed in all 14 tissues of STH sheep of the three FecB genotypes. BMPR1B was expressed in all selected tissues of the Han BB sheep, and was highly expressed in seven tissues (brain, cerebellum, hypothalamus, pituitary, ovary, uterus, and adrenal gland) of the Han B+ and Han ++ sheep. BMP15 was highly expressed in the ovaries of sheep of all three FecB genotypes. Additionally, GDF9 was expressed in all 14 tissues of all sheep.

Expression Levels of BMPR1B, BMP15, and GDF9 in the HPG Axis
It is very difficult to quantitatively analyze gene expression using RT-PCR. Therefore, the expression levels of BMPR1B, BMP15, and GDF9 in the HPG axis (hypothalamus, pituitary, and ovary), uterus, and oviduct tissues of the STH sheep were measured by qPCR.
As shown in Figure 3, BMPR1B was expressed in five tissues with the three FecB genotypes, with the highest level being in the ovaries (p < 0.01), followed by the hypothalamus, with no significant difference between the two. However, the expression level of BMPR1B in the hypothalamus was significantly higher than those of the pituitary, oviduct, and uterus (p < 0.01), and expression levels in the pituitary and oviduct were significantly higher than those of the uterus (p < 0.01, p < 0.05). In Han B+ and Han ++ sheep, the expression level of BMPR1B was significantly higher in the oviduct than in the pituitary (p < 0.01). There was no significant difference in the BMPR1B ovarian expression , and Han ++(c). 1-14: heart, liver, spleen, lung, kidney, brain, cerebellum, hypothalamus, pituitary, ovary, uterus, oviduct, adrenal gland, and duodenum, respectively. M: DL2000 DNA marker.

Expression Levels of BMPR1B, BMP15, and GDF9 in the HPG Axis
It is very difficult to quantitatively analyze gene expression using RT-PCR. Therefore, the expression levels of BMPR1B, BMP15, and GDF9 in the HPG axis (hypothalamus, pituitary, and ovary), uterus, and oviduct tissues of the STH sheep were measured by qPCR.
As shown in Figure 3, BMPR1B was expressed in five tissues with the three FecB genotypes, with the highest level being in the ovaries (p < 0.01), followed by the hypothalamus, with no significant difference between the two. However, the expression level of BMPR1B in the hypothalamus was significantly higher than those of the pituitary, oviduct, and uterus (p < 0.01), and expression levels in the pituitary and oviduct were significantly higher than those of the uterus (p < 0.01, p < 0.05). In Han B+ and Han ++ sheep, the expression level of BMPR1B was significantly higher in the oviduct than in the pituitary (p < 0.01). There was no significant difference in the BMPR1B ovarian expression levels between genotypes, but its expression in the hypothalamus of Han BB and Han B+ was higher than that in Han ++ sheep (p < 0.01, p < 0.05). BMPR1B expression levels in the pituitary and uterus were significantly higher in Han BB than in Han B+ and Han ++ sheep (p < 0.01, p < 0.05). However, BMPR1B expression in the oviduct was significantly higher in Han B+ than in Han BB and Han ++ sheep (p < 0.05).  The results of the analysis are shown in Figure 4, where among the five tissues examined, BMP15 was highly significantly expressed in the ovaries (p < 0.01) and presented in trace levels in nonovarian tissues. Additionally, the expression levels of BMP15 in the hypothalamus and oviduct of Han BB sheep were all much higher than those of the pituitary and uterus (p < 0.01), and expression in the pituitary was higher than in the uterus (p < 0.05). The tissue expression profiles of the three genotypes of sheep were analyzed, and the expression level of BMP15 in the ovaries of Han B+ sheep was significantly higher than in Han BB and Han ++ sheep (p < 0.05), and its expression in the ovaries of Han BB was lower than in Han ++ sheep; however, there was no significant difference between the two. Furthermore, the expression levels of BMP15 in the hypothalamus and oviduct tissues were much higher in Han BB than in Han B+ and Han ++ sheep (p < 0.05, p < 0.01).  The results of the analysis are shown in Figure 4, where among the five tissues examined, BMP15 was highly significantly expressed in the ovaries (p < 0.01) and presented in trace levels in non-ovarian tissues. Additionally, the expression levels of BMP15 in the hypothalamus and oviduct of Han BB sheep were all much higher than those of the pituitary and uterus (p < 0.01), and expression in the pituitary was higher than in the uterus (p < 0.05). The tissue expression profiles of the three genotypes of sheep were analyzed, and the expression level of BMP15 in the ovaries of Han B+ sheep was significantly higher than in Han BB and Han ++ sheep (p < 0.05), and its expression in the ovaries of Han BB was lower than in Han ++ sheep; however, there was no significant difference between the two. Furthermore, the expression levels of BMP15 in the hypothalamus and oviduct tissues were much higher in Han BB than in Han B+ and Han ++ sheep (p < 0.05, p < 0.01). Figure 5 clearly shows that the expression levels of GDF9 in the ovaries of sheep of the three FecB genotypes showed a similar pattern to BMP15, with its expression level being significantly higher in the pituitary than in the hypothalamus, oviduct, and uterus (p < 0.01). In addition, the expression level of GDF9 was significantly higher in the hypothalamus of Han BB and Han B+ sheep than in other tissues including the oviduct and uterus (p < 0.01). In the comparison between genotypes, the expression level of GDF9 in the ovaries was much higher in Han B+ than in Han BB and Han ++ sheep (p < 0.01 or p < 0.05), and its expression in the ovaries was higher in Han BB than in Han ++ shepp, but there was no significant difference between the two (p >0.05). In addition, GDF9 expression in the hypothalamus and pituitary was significantly higher in Han BB and Han B+ than in Han ++ sheep (p < 0.01, p< 0.05), and its expression in the uterus was much higher in Han BB than in Han ++ sheep (p < 0.05). in the pituitary was higher than in the uterus (p < 0.05). The tissue expression profiles of the three genotypes of sheep were analyzed, and the expression level of BMP15 in the ovaries of Han B+ sheep was significantly higher than in Han BB and Han ++ sheep (p < 0.05), and its expression in the ovaries of Han BB was lower than in Han ++ sheep; however, there was no significant difference between the two. Furthermore, the expression levels of BMP15 in the hypothalamus and oviduct tissues were much higher in Han BB than in Han B+ and Han ++ sheep (p < 0.05, p < 0.01).  value lower than 0.01, 0.05 are giving two asterisks (**) and one asterisk (*), respectively. Nonsignificant results are not giving any asterisks. Figure 5 clearly shows that the expression levels of GDF9 in the ovaries of sheep of the three FecB genotypes showed a similar pattern to BMP15, with its expression level being significantly higher in the pituitary than in the hypothalamus, oviduct, and uterus (p < 0.01). In addition, the expression level of GDF9 was significantly higher in the hypothalamus of Han BB and Han B+ sheep than in other tissues including the oviduct and uterus (p < 0.01). In the comparison between genotypes, the expression level of GDF9 in the ovaries was much higher in Han B+ than in Han BB and Han ++ sheep (p < 0.01 or p < 0.05), and its expression in the ovaries was higher in Han BB than in Han ++ shepp, but there was no significant difference between the two (p >0.05). In addition, GDF9 expression in the hypothalamus and pituitary was significantly higher in Han BB and Han B+ than in Han ++ sheep (p < 0.01, p< 0.05), and its expression in the uterus was much higher in Han BB than in Han ++ sheep (p < 0.05).

BMPR1B Expression
Previous reports have found that BMPR1B plays the same role as the protein kinase activity of serine or threonine, which has an important influence on ovarian biological function [32]. BMPR1B expression has previously been detected in the reproductive tissues, brain, skeletal muscle, and kidney tissues of sheep [11]. In this study, BMPR1B was shown to be highly and widely expressed in tissues, including the ovary and hypothalamus, in the three FecB genotypes of STH sheep. In Chinese Merino sheep, during the estrus period BMPR1B was shown to be expressed in the ovary, ear, spinal cord, pituitary, bone, uterus, hypothalamus, kidney, skeletal muscle, and oviduct tissues, which indicates that its function is diverse and that it plays a critical role in the ovaries in addition to bone formation [33]. However, the expression levels of BMPR1B in the ovarian tissues of the three FecB genotypes in Chinese Merino sheep ewes showed no significant difference [33]. Subsequently, BMPR1B expression in the follicles of multiparous Hu sheep was found to be significantly higher than that of uniparous Hu sheep, which indicated that it may influence the ovulation rate by regulating the BMP/Smad signal pathway and some related cytokines [34]. In addition, some researchers have found that BMPR1B has no tissue-specific expression in whole tissues, and it was

BMPR1B Expression
Previous reports have found that BMPR1B plays the same role as the protein kinase activity of serine or threonine, which has an important influence on ovarian biological function [32]. BMPR1B expression has previously been detected in the reproductive tissues, brain, skeletal muscle, and kidney tissues of sheep [11]. In this study, BMPR1B was shown to be highly and widely expressed in tissues, including the ovary and hypothalamus, in the three FecB genotypes of STH sheep. In Chinese Merino sheep, during the estrus period BMPR1B was shown to be expressed in the ovary, ear, spinal cord, pituitary, bone, uterus, hypothalamus, kidney, skeletal muscle, and oviduct tissues, which indicates that its function is diverse and that it plays a critical role in the ovaries in addition to bone formation [33]. However, the expression levels of BMPR1B in the ovarian tissues of the three FecB genotypes in Chinese Merino sheep ewes showed no significant difference [33]. Subsequently, BMPR1B expression in the follicles of multiparous Hu sheep was found to be significantly higher than that of uniparous Hu sheep, which indicated that it may influence the ovulation rate by regulating the BMP/Smad signal pathway and some related cytokines [34]. In addition, some researchers have found that BMPR1B has no tissue-specific expression in whole tissues, and it was also shown to have a high expression level in the ovaries of the Jining Grey goat and Liaoning Cashmere goat, implying that it not only exerts a great influence on the process of bone formation, but also has a potential role in ovarian development and even in the process of reproduction. However, no significant difference in expression was shown between these two goat breeds, which suggested that the BMPR1B may not contribute to the high fecundity of goats [35]. Similarly, it has also been speculated that differences in reproductive performance may be ascribed to the structural differences in BMPR1B between Lezhi black and Tibetan goats [36].
On the basis of the similarity of expression levels of ovarian BMPR1B in the three FecB genotypes, we propose that BMPR1B plays an important biological role in the lambing performance of STH sheep through mutation rather than expression level. Early studies found that granulosa cells (GC) may be inhibited by BMPR1B, but the inhibition effect of the mutated BMPR1B was weaker for GC. Sheep carrying the FecB mutation exhibit higher ovulation numbers [11], which may be the cause of the different litter sizes of sheep in the three FecB genotypes. Moreover, the expression differences in BMPR1B in non-ovarian tissues of the HPG axis of sheep of the three FecB genotypes were not consistent with the results from goat breeds [35]. We infer that the expression profile described above may have a species discrepancy or the different timings of the sampling may have influenced the results. Regarding BMPR1B expression in non-ovarian tissues, especially in the oviduct, previous research has suggested that its expression in the ampullary region of the bovine oviduct is significantly higher than in the isthmus area and may affect the normal physiological function of oviduct epithelial cells [37]. Therefore, we needed to explore whether the difference in expression of BMPR1B in non-ovarian tissues influences the ovulation rate and litter sizes of STH sheep of the three FecB genotypes. According to the analysis of function annotation and tissue-specific expression, especially regarding the high level of expression in the ovaries, the biological function of BMPR1B appears to be diverse. It can be speculated that BMPR1B exerts a pivotal influence on ovarian function, follicular development, and maturity.

BMP15 Expression
BMP15, which is secreted by oocytes, is an indispensable signaling molecule for normal follicular development [38] and can play a critical regulatory role in the growth and differentiation of early oocytes [39,40]. Studies have found that it can activate the mRNA transcription of PFKP (phosphofructokinase, platelet type) and lactate dehydrogenase A (LDHA), which are required for granulosa cell glycolysis [41]. Likewise, it can promote the expression of kit ligand mRNA in granulosa cells [42] and antagonize the apoptosis of cumulus cells [43]. Expression of BMP15 was first detected in mouse oocytes [44]. Subsequently, other researchers have found it to have high specific expression in human oocytes [17], as well as in rats [14], pigs [25], and sheep [45]. In this study, BMP15 was shown to be highly expressed in the ovaries, with trace expression in five other tissues (spleen, lung, kidney, hypothalamus, and oviduct), which is consistent with the results in Hu sheep [46]. Previous research indicated that BMP15 can downregulate the follicle stimulating hormone (FSH) effect, promote cell proliferation, and ensure the growth and maturity of oocytes by inhibiting the expression of the FSH receptor [35]. Therefore, BMP15, based on its high expression in the ovary, may be an important factor in maintaining numerous follicular developments.
In this study, BMP15 expression was lower in the ovaries of Han BB sheep than in Han B+ and Han ++ sheep. From previous studies, it is well-known that the expression level of BMP15 in the ovaries of FecB mutant homozygous (FecB BB genotype) Booroola Romney sheep is significantly lower than that in the wide-type (FecB ++ genotype), which suggests that the higher ovulation rate of BB genotype sheep is due to a decrease in the BMP15 mRNA level and an earlier onset of luteinizing hormone (LH)-responsiveness in granulosa cells (GC) [45]. The receptor concentration of synthetic estrogen, inhibin, and follicle stimulating hormone (FSH) in follicular granulosa cells decreases due to whole or partial deletion of BMP15 function, which further leads to lower levels of FSH that maintain the follicular growth. Therefore, it can be inferred that ewes with the FecB mutant have a high ovulation rate, which may be responsible for the low expression of BMP15 in the ovaries of Han BB sheep. However, some studies have also concluded that BMP15 is a key gene in the high fecundity of goats based on results where BMP15 expression in the hircine ovary was shown to be significantly higher in a polytocous breed than in a monotocous breed [35,47]. These studies suggest that BMP15 has a substantial regulatory effect on the ovulation rates of sheep and goat [48,49]. The high level expression of BMP15 in the ovaries of Han B+ sheep, implies that BMP15 may in addition to its demonstrated function in the ovary of FecB B+ genotype STH sheep, have other influences on reproduction. For BMP15, in regard to the different expression levels in non-ovarian tissues, we hypothesize that unclear effects related to production or interrelationships with other genes could lead to this expression profile. Generally speaking, the high expression of BMP15 in the ovaries indicates that it plays a key role in the growth and maturation of oocytes. However, its expression level may be negatively correlated with the litter sizes of STH sheep.

GDF9 Expression
GDF9 is secreted by oocytes and performs pivotal regulation in follicular growth and differentiation by means of the paracrine pathway [50]. A previous study reported that GDF9 promotes the expression of hyaluronan synthase 2 and urokinase plasminogen activator (UPA) in the cumulus oocyte complex (COC) and activates the cumulus expansion [51]. GDF9 and its protein have been expressed in many mammal oocytes via in situ hybridization and immunohistochemical technologies [52][53][54]. GDF9 was also detected in GC [18] and non-ovarian tissues, including the hypothalamus pituitary and testis [26]. Particularly in Hu sheep, it is expressed in 10 tissues (hypothalamic, pituitary, ovary, oviduct, uterus, heart, liver, spleen, lung, and kidney tissues) [55], which implies that GDF9 has a wide range of biological effects. In our study, GDF9 was expressed in all tissues, of which the ovaries showed the highest expression, which indicates that it plays an important part not only in reproductive organs, but also in other organs and tissues.
Additionally, previous studies revealed that GDF9 expression in the ovaries is significantly higher in prolific Hu sheep than in non-prolific Hu sheep, indicating that it is a differentially expressed gene and could also affect the BMP/Smad metabolic pathway to regulate the litter sizes of sheep [34]. Previous research has revealed that GDF9 can exert a synergistic effect with FSH, BMP15, and other hormones or growth factors during follicular development [18]. In this study, the GDF9 expression levels in the ovaries were higher in STH sheep with Han BB and Han B+ genotypes than in those with Han ++ genotype, which indicates that GDF9 expression may affect the lambing performance of different FecB genotypes of STH sheep. However, this is an inconsistent conclusion as the GDF9 expression level in the ovaries was not significantly different between FecB carrier and noncarrier Indian sheep [56]. It was also found that the expression levels of GDF9 in the HPG axis tissues of the Jining Grey goat (multiparous breed) and Liaoning Cashmere goat (uniparious breed) are not significantly different [35]. These different findings may be the result of differences between species or breeds and the timeliness of sampling. As a consequence, GDF9 may implement a similar function in these tissues to promote oocyte maturity and further affect the ovulation rate in STH sheep. Therefore, we concluded that GDF9 function is complex and plays a positive regulatory role in the differentiation and development of antral follicles, eventually possibly increasing the ovulation rate and litter size of STH sheep with the FecB mutation.

Conclusions
This study found that BMPR1B, BMP15, and GDF9 are highly expressed in the ovaries of three FecB genotypes of STH sheep, which indicates that they may play pivotal functions in the ovine ovaries and promote follicular growth and maturation. The BMPR1B expression level may not differ significantly in the ovaries of STH ewes of the three genotypes, while BMP15 and GDF9 genes have an important biological function in the ovary and influence the reproduction performance of FecB B+ genotype STH sheep. This is the first systematic analysis of tissue expression patterns of the three genes tissue expression pattern in FecB genotypes of STH sheep.