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. 2020 Apr 17;98(5):skaa100. doi: 10.1093/jas/skaa100

Study on the correlation between BMPR1B protein in sheep blood and reproductive performance

Xiaoyan Zhang 1, Liping Zhang 1,✉, Weibo Sun 1, Xia Lang 2, Jianping Wu 2, Caiye Zhu 1, Jianlei Jia 2, Jinpen Jin 3, Younfu La 1, David P Casper 4
PMCID: PMC7199883  PMID: 32300800

Abstract

Sheep reproductive performance is one of the important economic traits in sheep farming. The bone morphogenetic protein receptor 1B (BMPR1B) gene and protein may play an important role in sheep fertility. This study was to investigate the association of blood BMPR1B protein expression with reproductive performance in sheep. Mongolian sheep with single and twin births and polytocous Small Tail Han sheep were selected due to differences in birth numbers. The BMPR1B mRNA in sheep blood was measured by a reverse transcription-polymerase chain reaction as well as the BMPR1B protein was measured by enzyme-linked immunosorbent assay in blood samples of Mongolian and Small Tail Han sheep. The results demonstrated that blood BMPR1B concentration in Mongolian sheep with twin birth was higher (P < 0.05) than Small Tail Han sheep and Mongolian sheep with single birth. The protein concentration in the anestrus season was higher (P < 0.045) than those in the estrus season for both Mongolian and Small Tail Han sheep. Moreover, BMPR1B concentration in Mongolian sheep increased (P < 0.05) at the age of 6 to 12 mo and that in Small Tail Han sheep increased (P < 0.05) at the age of 3 to 6 mo. The result indicates that the increase in BMPR1B protein concentrations in the blood of Mongolian ewes and Small Tail Han ewes may be beneficial to follicular development, but too high or too low of this blood protein concentration in Mongolian and Small Tail Han sheep is not conducive to ovulation.

Keywords: bone morphogenetic protein receptor 1B protein, Mongolian sheep, reproductive performance, Small Tail Han sheep, sheep blood

Introduction

Sheep reproductive performance is an important economic trait for the sheep industry. Sheep fertility has complex molecular regulatory mechanisms and the regulatory mechanism controlling sheep reproductive performance is an exciting research area. Bone morphogenetic protein receptor 1B (BMPR1B), also known as activin-like kinase 6, is a member of the bone morphogenetic protein (BMP) receptor family (Edson et al., 2010). The transmembrane protein produced by the BMPR1B gene mainly participates in the signal transduction pathway of transforming growth factor-beta superfamily to promote ovarian follicular and embryonic development (Cao and Chen, 2005; Gazzerro and Canalis, 2006; Alsamerria et al., 2015).

The ovine BMPR1B gene is located on chromosome 6, which consists of 12 exons (1,509 bp) and encodes 502 amino acids (Pan et al., 2015a). There are more than 20 single nucleotide polymorphisms (SNPs) available for the sheep BMPR1B gene (Chu et al., 2011), among which c.746A > G, c. 864 T > C, and c. 1,113 C > A are associated with sheep reproduction (Du et al., 2017; Ma et al., 2017). A major research on the non-synonymous SNP of c.746A > G found that the damage to the BMP system during follicle development led to increase average ovulation in Australian Booroola Merino (Mulsant et al., 2001), Small Tail Han sheep (Chu et al., 2007), and Hu sheep (Wang et al., 2003). At the same time, this gene has an additive effect on litter and ovulation rate but has negative effects on fetal growth and development and body mass during gestation (Gootwine et al., 2006). The BMPR1B gene is considered a possible candidate gene for regulating sheep reproduction (Chu et al., 2011. The gene is widely expressed in sheep hypothalamus, pituitary, uterus, and ovary (Tang et al., 2018). Sun et al. (2010) had detected BMPR1B gene expression in adult mice and porcine follicles by immunohistochemistry (Sun et al., 2010). Yi et al. (2001) reported that BMPR1B-deficient mice might develop irregular estrus cycles and reproductive problems, such as pseudopregnancy (Yi et al., 2001). However, there is little information evaluating the BMPR1B mRNA and protein expression in sheep peripheral blood.

The hypothesis is that the BMPR1B gene plays a crucial role in regulating sheep reproduction relating to multiple births. Mongolian sheep having single and twin births and polytocous Small Tail Han sheep were selected to evaluate BMPR1B gene impacts on reproductive cycling. The expression of the BMPR1B gene mRNA and protein concentration were measured using reverse transcription-polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) methods in collected Small Tail Han sheep and Mongolian sheep blood samples. This study could provide critical information for the regulatory mechanism of litter size, sexual maturity, and seasonal estrus in sheep.

Materials and Methods

Animal care

Animal care and experimental procedures were approved by the Animal Committee Guidelines of the Gansu Agricultural University (Lanzhou, China) and the National Natural Science Foundation of China according to guidelines established by the Biological Studies Animal Care and Use Committee of Gansu Province (Approval No. 31660670), which follows the guidelines of the Chinese Standards for the Use and Care of Research Animals (He et al., 2016).

Animal samples

The experimental investigation started with a total of 128 female sheep sourced from the Hexi region in Gansu province, China. Four adult Mongolian breeding ewes and four Small Tail Han breeding ewes were selected from the flock to examine the initial expression of BMPR1B mRNA in sheep blood. To determine BMPR1B mRNA and protein concentrations and possible cycling patterns that could be associated with the reproductive cycle of ewes, blood samples were collected from ewes demonstrating different reproductive performances. Specifically, 3- to 4-yr-old polytocous Small Tail Han sheep (n = 10 with average litter size ≥ 3) and Mongolian sheep demonstrating only a single birth (n = 10 with litter size = 1) and Mongolian sheep demonstrating twin births (n = 10 with litter size = 2) were selected for blood sampling. All ewes were blood sampled in both the estrus season (November, Autumn) and the anestrus season (April, Spring) for comparing BMPR1B concentrations during nonreproductive and reproductive cycles.

To ascertain if female lamb age could also be an influencing factor on BMPR1B protein concentrations, at each age group, Small Tail Han sheep aged 3 mo old (n = 10), 6 mo old (n = 10), and 12 mo old (n = 10) along with Mongolian sheep aged 3 mo old (n = 10), 6 mo old (n = 10), and 12 mo old (n = 10) in April were selected for blood sampling. The fecundity Booloola (FecB) genotypes of all ten 3- to 4-yr-old Small Tail Han sheep were determined to be homozygous mutant BB (highest ovulation rate, Guo et al., 2018) as evaluated by PCR, DNA sequencing, and three lambing records per ewe (Du, 2018). All twenty 3- to 4-yr-old ewes were in the empty period (nonpregnant). All selected sheep for this experiment were deemed healthy, free of reproductive diseases, and received no hormonal treatments.

Blood sample collection

Blood samples were collected via 10-mL Vacutainer tubes with a 18-guage needle (Becton, Dickinson and Company, Franklin Lake, NJ). Whole blood was collected from the jugular vein from four Mongolian and four Small Tail Han sheep approximately 4 yr of age in April. These 4-yr-old ewes were randomly selected from the larger flock at the sheep farm. Whole blood was collected and stored using cryotubes (Corning, New York). Tubes contained ethylenediaminetetraacetic acid, added RNALock blood RNA stabilizer (DP440, Tiangen, Beijing, China), and was immediately snap-frozen in liquid nitrogen, transported back to the laboratory, and stored at −70 °C for later analyses of total RNA.

Blood samples for the measurement of BMPR1B protein concentrations were collected from the jugular vein of each sheep via a 10-mL Vacutainer tube without anticoagulant using a 18-gauge needle (Becton, Dickinson and Company) at 7:30 a.m. before the morning feeding. Blood tubes were centrifuged at 2,000 × g for 20 min at 4 °C, serum was collected, and stored in 1.5 mL centrifuge tubes (Corning, New York), and frozen at −20 °C until assayed for BMPR1B concentrations. Sample storage and laboratory assays were scheduled to avoid subjecting samples to freeze-thaw cycles.

Blood mRNA isolation and BMPR1B mRNA detection by reverse transcription-PCR

Total RNA from blood serum samples was isolated using the RNAprep pure blood kit (DP433, Tiangen, Beijing, China), according to the manufacturer’s instructions. The total RNA was produced using the reverse transcribed two-step method of the Primescript RT Reagent Kit with gDNA Eraser (Takara, Dalian, China). The reverse transcription product was stored in a −20 °C freezer (Haier, Qingdao, China).

The primers used were designed to amplify a 167-bp fragment of the BMPR1B (GenBank No. NM_001009431.1) gene using reverse transcription-PCR. All primers were synthesized by GENEWIZ Biotechnology Co., Ltd (Suzhou, China). The forward primer is 5′-GCTGGTTCCGAGAGACAGAAA-3′ and the reverse primer is 5′-GGTGGTGGACTTCAGGTAATCATAG-3′.

The PCR reaction was conducted using a 20-uL volume with a reaction mixture containing 10 μL TaqMix (Real-Times, Beijing, China), 7 μL H2O, 1 μL of forward and reverse primers, and 1 μL cDNA. The PCR procedure was conducted under the following thermal conditions: initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 30 s, with a final extension at 72 °C for 10 min. The PCR amplification products were detected using agarose gel electrophoresis. The separated PCR amplification products were sequenced at GENEWIZ Biotechnology Co., Ltd (Suzhou, China).

Blood BMPR1B protein detection by ELISA

The sheep blood serum BMPR1B protein concentrations were measured by conducting duplicated analyses on each sample using a commercial ELISA Kit (Sheep Bone Morphogenetic Protein Receptor 1B ELISA Kit, Cat. # EIA-10072, Elisa Biotech Co., Ltd., Shanghai, China) according to the manufacturer’s instructions. The assay sensitivity was approximately less than 0.1 ng/mL with a linear range of 0.625 to 20 ng/mL. Intra- and inter-assay coefficient of variations (%) were less than 15%. The assay can recognize both recombinant and natural sheep BMPR1B protein with no significant cross-reactivity or interference. The standard curve concentrations (i.e., 0.625, 1.25, 2.50, 5.00, 10.00, and 20.00 ng/mL) provided by the manufacturer were used to establish the standard curve. Optical density (absorbance) was measured at 450 nm using a SpectraMax Absorbance Plate Reader CMax Plus (Molecular Devices, Sunnyvale, USA).

Statistical analysis

Sequence alignments were constructed using MEGA 5.0 (Arizona State University, AZ, USA) and NCBI Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Statistical data were analyzed using SPSS version 19.0 (IBM Inc., Armonk, NY, USA). All data were expressed as means ± standard error. One-way ANOVA comparisons (Steele and Torrie, 1980) were conducted and the Student’s t-test (least significant difference) was used to separate treatment means. Significant differences between means were declared when P was <0.05.

Results

BMPR1B mRNA expression

The BMPR1B amplification products produced via reverse transcription-PCR when separated via electrophoresis on 1.2% agarose gel demonstrated fragment lengths consistent with theoretical lengths (Figure 1). As demonstrated in Figure 1, the BMPR1B gene is well expressed in Mongolian and Small Tail Han sheep blood. The sequencing results (File S3) demonstrated a 100% alignment when evaluating sequences using MEGA 5.0 and NCBI blast software between the amplified BMPR1B cDNA fragment and the published reference sequence (NM_001009431.1, GenBank).

Figure 1.

Figure 1.

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The expression of BMPR1B mRNA in sheep blood. M: 2,000 base pairs (BP) DNA marker; 1, 2: Mongolia sheep with single birth; 3, 4: Mongolia sheep with twin birth; 5, 6: Polytocous Small Tail Han sheep.

ELISA test results

The standard curve demonstrated an excellent linear relationship between standard concentrations and optical density (absorbance, A = 12.83X + 0.074; R2 > 0.99) to be able to measure and calculate blood BMPR1B protein concentrations.

Sheep blood BMPR1B protein concentrations with different reproductive performance

During the anestrus season, low-fecundity, mature Mongolian (3- to 4-yr old) ewes demonstrated lower (P < 0.05) BMPR1B protein concentrations compared with high-fecundity Mongolian twin birthing or polytocous Small Tail Han mature sheep (Table 1). However, blood BMPR1B protein concentrations were similar for the Mongolian ewes and Small Tail Han sheep (i.e., breed). In contrast, during the estrus season, Mongolian sheep having twin births demonstrated greater (P < 0.05) blood BMPR1B protein concentrations compared with Mongolian having a single birth (lamb) and polytocous Small Tail Han sheep. The blood BMPR1B protein concentration was similar (P > 0.05) but numerically greater for polytocous Small Tail Han sheep compared with Mongolian sheep when both breeds gave birth to a single lamb.

Table 1.

Blood BMPR1B protein concentrations for polytocous Small Tail Han Sheep (PSTHS), Mongolian Sheep with Twin Births (MSWTB), or Mongolian Sheep with Single Birth (MSWSB) with different reproductive performance and at different ages (ng/mL)

Breed
Season N PSTHS MSWTB MSWSB
Estrus season 10 3.17 ± 0.06b 3.80 ± 0.22a 2.90 ± 0.09b
Anestrus season 10 11.15 ± 0.78 11.25 ± 0.91 9.58 ± 0.73
Age, months
Breed N 3 6 12
Small Tail Han sheep 10 2.60 ± 0.35b 10.57 ± 0.78a 9.97 ± 0.63a
Mongolian sheep 10 8.26 ± 0.70b 7.16 ± 0.42b 13.01 ± 1.38a
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a,bMeans within the same row with different superscripts differ, P < 0.05.

Sheep blood BMPR1B protein concentrations in estrus and anestrus season

Seasonally, both sheep breeds (Mongolian and Small Tail Han) responded similarly (P > 0.05) in that the BMPR1B protein concentrations are higher (P < 0.05, in anestrus compared with estrus seasons (Table 1).

Sheep blood BMPR1B protein concentrations at different ages

The blood serum BMPR1B protein concentration in 12-mo-old Mongolian sheep was greater (P < 0.05) compared with Mongolian sheep at 3 and 6 mo of age (Table 1). In contrast to the Mongolian sheep, the Small Tail Han sheep demonstrated higher (P < 0.05) BMPR1B protein concentrations at 6 and 12 mo compared to 3 mo of age. The young female lambs have greater (P < 0.05) concentrations of BMPR1B protein at 1 yr of age compared with mature sheep during the estrus season, while being similar (P > 0.05) to the higher concentrations occurring during the anestrus seasons, which could imply that BMPR1B protein is inhibiting estrus in young growing female lambs.

Blood serum BMPR1B protein concentrations appear to change with age (Table 1). These data demonstrate at 3 mo of age BMPR1B protein concentrations are greater (P < 0.05) for Mongolian sheep compared with Small Tail Han sheep; however, this observation reverses for 6 mo of age. At 12 mo of age, BMPR1B protein concentrations were greater (P < 0.05) for Mongolian compared with Small Tail Han sheep. These observations may be related to the fecundity of the sheep breeds

Discussion

The BMPR1B gene encodes the BMPR1B protein that contains three parts: the N-terminal extracellular ligand-binding domain, the single transmembrane region, and the C-terminal serine/threonine kinase domain (Mulsant et al., 2001; Souza et al., 2001; Lima et al., 2012). After binding with BMP ligands, BMPR-IIB transphosphorylates the Gly-Ser domain of BMPR1B, leading to activation of biological responses including reproductive function (Mulsant et al., 2001; Kaivo-Oja et al., 2006). Phosphorylated BMP type 1 receptors can induce phosphorylation of receptor-specific Sma- and mothers against decapentaplegic (SMAD) and form oligomeric complexes with common partner SMAD (Kaivo-Ojaq et al., 2006). This complex translocates to the nucleus to regulate specific gene expression, causing various biological responses including reproductive function (Kaivo-Oja et al., 2006). Recently, Abdurahman et al. (2019) reported that SMAD4 feedback enhances BMPR1B transcription in ovine granulosa cells.

The BMPR1B gene affects ovine ovulation rate, litter size, and growth traits in sheep (Gootwine et al., 2006). These results suggest that changes in blood BMPR1B concentrations in Mongolian sheep may be related to ovulation during the estrus season. This study reports that the blood serum BMPR1B protein concentrations of Mongolian sheep having twins and polytocous Small Tail Han sheep were greater than Mongolian sheep with single births. The BMPR1B mRNA was found to be more abundant in the antral follicles of high-fecundity Hu sheep than those of low-fecundity Hu sheep (Xu et al., 2010). The expression of BMPR1B mRNA in the estrus of Mongolian sheep was 2.65 times higher than that in the anestrus (He et al., 2010). In ovary, hypothalamus, pituitary, and uterus, there was no difference in the BMPR1B gene expression level between prolific Jining Gray goat and non-prolific Liaoning Cashmere goats. Additionally, the BMPR1B gene expression level was the highest in the ovary (Pan et al., 2015b). The metabolic effects of the BMPR1B gene are more pronounced in follicular than in ovarian vein serum in Small Tail Han sheep, and part of the follicular fluid is the transfer of plasma components across the blood follicle barrier (Guo et al., 2018). The Small Tail Han sheep breed is known worldwide for being hyperprolificacy, sexual precocity, and year-round estrus. In contrast, the Mongolian sheep breed is non-prolific by usually producing one lamb each birth and seasonal breeding (Zhao, 2011; Wang, 2012; Zhao, 2013). Luong et al. (2011) reported that rare BMPR1B variants may still contribute to human dizygotic twinning. In addition, BMPR1B is associated with skeletal development (Yoon et al., 2005), pathogenesis of idiopathic pulmonary hypertension in children (Chida et al., 2012), migration of breast cancer (Allison et al., 2016), and inhibition of ovarian cancer (Chu et al., 2007).

Sheep generally reach puberty (initiate estrus cycling) at 4 to 5 mo of age but the female reproductive organs are still gradually growing to reach sexual maturity at 6 to 10 mo (Zhao, 2011) with a normal estrus cycle and ovulation for normal reproductive capacity (Wang, 2012). However, Small Tail Han sheep generally reach sexual maturity at 5 to 6 mo of age, while Mongolian sheep reach sexual maturity later at 7 to 8 mo of age (Zhao, 2011). To confirm these observations, it was found that blood BMPR1B protein concentrations in Mongolian sheep increased significantly at 6 to 12 mo of age, while for Small Tail Han sheep BMPR1B concentrations increased significantly earlier at 3–= to 6 mo of age. The age when blood BMPR1B protein concentrations in Mongolian and Small Tail Han sheep increased in association (correlated) with sexual maturity. The speculation is that elevated blood BMPR1B protein concentrations in Mongolian and Small Tail Han sheep may be related to sexual maturity and follicular development.

Seasonal estrus is a critical factor limiting sheep fecundity and productivity due to changes in both ovarian function and hormonal secretions during the different seasons. Seasonal sheep breeding is controlled by factors, especially the annual photoperiodic cycle, which causes changes in reproductive hormonal secretions leading to estrus (Smith and Clark, 2010). Thus, ovarian follicular development demonstrated by seasonal breeding sheep is experienced by annual periods of quiescence and renaissance. During anestrus, ovarian physiological activity is reduced with the follicular stage not developing (Di et al., 2014). During the selection process of the dominant follicle, the expression of BMPR1B protein in the follicular granule cells declined followed by an increase in follicle size (Regan et al., 2015). Granular cells in atretic cells continued to demonstrate a high expression of BMPR1B mRNA (Regan et al., 2016). This study found that blood BMPR1B protein concentrations during the anestrus season were significantly higher than during the estrus season in both Mongolian and Small Tail Han sheep breeds. Regan et al. (2016) reported that the dysregulation of granulosal BMPR1B protein density is associated with decreased ovarian reserve and an age-related decline in human fertility. This study found significantly higher blood BMPR1B protein concentrations in both Mongolian and Small Tail Han adult ewes during the anestrus compared with estrous seasons. It is speculated that changes in blood BMPR1B protein concentrations may be affected by seasonal factors, therefore influencing reproductive performance. In contrast, Mongolian sheep known as a seasonal estrous sheep demonstrated significant changes in blood BMPR1B protein concentrations during the estrous and anestrus season, suggesting that blood BMPR1B protein concentrations may be associated with seasonal estrus. Furthermore, these findings suggest that the increase in blood BMPR1B protein concentrations for both Mongolian and Small Tail Han sheep ewes may be beneficial to follicular development. However, too high or too low of blood BMPR1B protein concentrations are not conducive to ovulation.

Conclusions

In this study, it was found that the expression of BMPR1B mRNA and associated blood protein concentration in different ages and reproductive periods of Mongolian and Small Tail Han Sheep are associated with the estrus and anestrus cycling of the reproductive phases. Blood BMPR1B protein concentration for both Mongolian sheep with twin lambs and Small Tail Han sheep with multiple births were higher than Mongolian sheep with single births. Blood BMPR1B concentrations during the anestrus season were significantly higher than during the estrus season for both Mongolian and Small Tail Han sheep. In addition, blood BMPR1B protein concentrations for young growing Mongolian and Small Tail Han sheep significantly increased during the period of 6 to12 mo of age compared with 3 mo of age. These data confirm that the BMPR1B gene and its blood protein concentrations are associated with reproductive performance in sheep.

Acknowledgment

This experiment was funded by the Chinese National Natural Science Foundation (31260547), the Gansu Agricultural University Fund Project (GSAU-ZL-2015–027), and “Research and demonstration on nutrition balance of grazing cattle and sheep” special scientific research project of public welfare industry (Agriculture) (201303062).

Glossary

Abbreviations

BMP

bone morphogenetic protein

BMPR1B

bone morphogenetic protein receptor 1B

ELISA

enzyme-linked immunosorbent assay

MRA

messenger ribonucleic acid

PCR

polymerase chain reaction

SMAD

Sma- and mothers against decapentaplegic

SNP

single nucleotide polymorphism

Conflict of interest statement

The authors declare no real or perceived conflicts of interest. The funding agencies did not participate in study design, data collection, analysis, and interpretation or writing of the manuscript.

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