Expression Analysis of BMPR1B, BMP15, GDF9, Smad1, Smad5, and Smad9 in Rams with Different Fecundity

Weihao Chen1,2, Zhilong Tian2, Lin Ma2, Shangquan Gan3, Wei Sun1,4,* and Mingxing Chu2,*

1College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China

2Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Agriculture, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China

3State Key Laboratory for Sheep Genetic Improvement and Healthy Production, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China

4Joint International Research Laboratory of Agriculture and Agri-Product Safety, Ministry of Education, Yangzhou University, Yangzhou 225009, China

ABSTRACT

To elucidate the tissue expression levels of BMPR1B, BMP15, GDF9, Smad1, Smad5, and Smad9 genes in rams with different fecundity, quantitative real-time polymerase chain reaction was used to investigate the expression level of six genes in the brain, cerebellum, hypothalamus, pituitary, testis, epididymis, vas deferens, and adrenal gland in high fecundity (Small Tail Han sheep) and low fecundity (Sunite sheep) rams. The results were as follows: BMPR1B, GDF9, Smad1, Smad5 and Smad9 were expressed in all selected tissues, but BMP15 was specifically expressed in the epididymis. Further study indicated that the expression of BMPRIB in the brain, hypothalamus, pituitary, epididymis, and adrenal gland was significantly higher in Sunite sheep than in Small Tail Han sheep (p < 0.05, p < 0.01); the expression of BMP15 in the epididymis was significantly higher in Sunite sheep than in Small Tail Han sheep (p < 0.01); the expression of GDF9 in the cerebellum and vas deferens was significantly higher in Small Tail Han sheep than in Sunite sheep (p < 0.05); the expression of GDF9 in the adrenal gland was significantly higher in Sunite sheep than in Small Tail Han sheep (p < 0.01); the expression of Smad1 in the brain and adrenal gland was significantly higher in Small Tail Han sheep than in Sunite sheep (p < 0.05); the expression of Smad1 in vas deferens was significantly higher in Sunite sheep than in Small Tail Han sheep (p < 0.01); the expression of Smad5 in the adrenal gland was significantly higher in Small Tail Han sheep than in Sunite sheep (p < 0.05); the expression of Smad9 in the brain and epididymis was significantly higher in Sunite sheep than in Small Tail Han sheep (p < 0.05, p < 0.01); and the expression of Smad9 in the cerebellum and hypothalamus was significantly higher in Small Tail Han sheep than in Sunite sheep (p < 0.05). This is the first study to systematically analyze the BMPR1B, BMP15, GDF9, Smad1, Smad5, and Smad9 genes’ tissue expression pattern in rams.

Article Information

Received 26 February 2019

Revised 11 May 2019

Accepted 11 June 2019

Available online 06 May 2020

Authors’ Contribution

WH-C, ZL-T, L-M and SQ-G designed the experiments and analysed the data. WH-C and W-S wrote the manuscript. MX-C supervised the research and approved the experiment.

Key words

Ram, BMPR1B, BMP15, GDF9, Smad, Tissue expression.

DOI: https://dx.doi.org/10.17582/journal.pjz/20190226030219

* Corresponding authors: mxchu@263.net;

dkxmsunwei@163.com

0030-9923/2020/0005-1665 $ 9.00/0

Copyright 2020 Zoological Society of Pakistan

Introduction

Lambing is one of the most economically important traits of sheep, being closely related to the economic benefits of sheep breeding. The major genes related to the prolificacy of sheep have received much attention from researchers since the 1980s (Tang et al., 2018). To date, three major candidate genes for the prolificacy of sheep have been found: bone morphogenetic protein receptor 1B (BMPR1B), bone morphogenetic protein 15 (BMP15), and growth differentiation factor-9 (GDF9) (Pan et al., 2018).

BMPR1B is the first major candidate gene found to be related to the prolificacy of sheep (Shokrollahi et al., 2018) and possesses a mutation (A to G), known as FecB, which results in one amino acid substitution (Q to R) increasing the ovulation rate of Booroola ewes (Montgomery et al., 1994). The FecB mutation has an additive effect on ovine ovulation number and litter size, so that one copy of the FecB mutation can increase the ovulation number by 1.3–1.6-fold and the litter size by 0.9–1.2-fold, and two copies by 2.73 and 1.1–1.7, respectively (El-Seedy et al., 2017).

BMP15 also known as growth differentiation factor-9B (GDF9B), and GDF9, which both belong to the transforming growth factor-β (TGF-β) superfamily and are recognized as major candidate genes for the prolificacy of sheep—were found to regulate the growth and differentiation of follicles, the secretion of reproductive hormones, and the growth of germ cells (Belli et al., 2018). To date, FecXI, FecXH (Galloway et al., 2000), FecXG (Hanrahan et al., 2004), FecXL, FecXB (Bodin et al., 2007), FecXR (Monteagudo et al., 2009), FecXGr , FecXO (Julie et al., 2013) and FecXBar (Lassoued et al., 2017) mutations have been found on the sheep BMP15 gene; they strong affect the ovulation rate and prolificacy, similar to the effect of G1 (Wang et al., 2018), G4 (Alam et al., 2018), G6, FecGF (G7) (Våge et al., 2013), FecGH (G8) (Shafieiyan et al., 2013), FecGE (Silva et al., 2011), FecTT (FecI) (Braun et al., 2003), FecGV (Souza et al., 2014), FecGT (Nicol et al., 2009),and FecGSI (Mullen et al., 2014) mutations on the sheep GDF9 gene.

Many studies revealed that the prolificacy of sheep is closely related to bone morphogenetic proteins (BMPs), BMP receptors (BMPR), and Smads, a downstream signaling molecule of the TGF-β/Smad signaling pathway (Lin et al., 2018). Members of the BMPs initiate signaling from the cell membrane by interacting with two distinct serine/threonine kinase receptors. Ligand binding induces the formation of a complex in which the type II receptor phosphorylates and activates the type I receptor. This protein then propagates the signal by phosphorylating the Smad proteins such as Smad1, Smad5, and Smad9 (Song et al. 2018). Phosphorylated Smad1/5/9 can form a complex by interacting with Smad4, which can further activate or inhibit the expression of target genes (Rol et al., 2018). Given its interaction with BMPs, Smad1/5/9 might be related to the prolificacy of sheep.

Small Tail Han sheep (STH) and Sunite sheep (SNT) are two Chinese local sheep (Ovis aries) breeds with different estrous modes (year-round and seasonal, respectively. Both are known for their excellent meat production performance (Tang et al., 2018). Significant differences between the two sheep breeds in fecundity have resulted in increasing interest in the expression pattern of major prolificacy genes in these sheep.

BMPR1B, BMP15, GDF9, Smad1, Smad5, and Smad9 are important in prolificacy. Many studies on the expression of these six genes in the tissues of ewes have been reported; however, no research has yet been reported about these genes in rams. To explore the potential role of these six genes in rams, we analyzed the tissue expression profile and the mRNA expression levels in eight prolificacy-related tissues between high fecundity sheep breed (STH) and low fecundity sheep breed (SNT) rams. Our study helps elucidate the genetic mechanism controlling high fecundity in rams.

 

Materials and Methods

Selection of experimental sheep and sample collection

The 3 Small Tail Han rams and 3 Sunite rams were supplied by Yuncheng Breeding Sheep Farm (Yuncheng County, China) and Sheep and Goat Breeding Farm of Tianjin, Institute of Animal Sciences (Tianjin, China). All rams were healthy, approximately 2.5 years old, and were kept in a sheltered outdoor paddock and were provided with alfalfa hay and concentrate, with clear water available ad libitum. Eight tissues (brain, cerebellum, hypothalamus, pituitary, testis, epididymis, vas deferens, and adrenal gland) were collected from each animal. All tissues were snap-frozen in liquid nitrogen and then stored at −80°C to be used for RNA extraction.

All experimental procedures mentioned in the present study were approved by the Science Research Department (in charge of animal welfare issue) of the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences (IAS-CAAS) (Beijing, China). Ethical approval was provided by the animal ethics committee of IAS-CAAS (No. IASCAAS-AE-03, December 12, 2016).

Total RNA extraction and cDNA synthesis

Tissue RNA was extracted from the 8 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 (each tissue smashed, mixed, and 50–100 mg used for RNA extraction). The quantity and quality of total RNA were monitored using 1.5% agarose gel electrophoresis and ultraviolet spectrophotometry (UV-1201, Shimadzu, Kyoto, Japan), respectively. Then, the RNA was stored at −80°C until use.

The first strand of cDNA was prepared following the instructions of the PrimeScriptTM 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 sec, with a total volume of 20 μL that contained PrimeScript RT Enzyme 1.0 µL, Random 6 mers 1.0 µL, 5 × PrimeScript Buffer (for Real Time) 4.0 µL, total RNA 1.0 µL and RNase-free ddH2O 13 µL. Prior to storage at −80°C, the standard working concentration of cDNA is 200ng/ul, the cDNA quality was evaluated by housekeeping gene (RPL-19) amplification, and then the reverse products were stored at −20°C until use.

RNA extraction and cDNA synthesis

Total RNA samples were analyzed using 1.5% agarose gel electrophoresis (U = 150 V; I = 240 mA). Three bands were detected (28S, 18S, and 5S)—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 RNA samples 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 were appropriate for use in the follow-up experiment.

Primer design

A total of 7 primers were designed using the Primer Premier (version 5.0, PREMIER Biosoft Co., Palo Alto, CA, USA) software to amplify different fragments of ovine BMPR1B, BMP15, GDF9, Smad1, Smad5, Smad9, and RPL-19 genes based on their assembled sequences in GenBank. All primers were synthesized by Beijing Tianyi Biotechnology Co., Ltd. (Beijing, China). The housekeeping gene (RPL-19, accession number: XM_012186026.1) was used as an internal control to normalize the threshold cycle (Ct) values. The primers are detailed in Table I.

qPCR

Real-time polymerase chain reaction (PCR) amplification was performed in 20 μL of reaction mixture that contained 10 µL SYBR Premix EX Taq II (TaKaRa Bio Inc., Dalian, China), 0.4 µL of each forward and reverse primer, 6.4 µL RNase-Free ddH2O, and 2 µL cDNA. PCR amplification was performed in triplicate wells using the following conditions: initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 10 sec, and 60°C for 30 sec. The dissociation curve was analyzed after amplification. A melting temperature (Tm) peak at 85±0.8°C on the dissociation curve was used to determine the specificity of PCR amplification.

Data

The 2−ΔΔCt method (Guo et al. 2018) was used to process the real-time PCR results. Statistical analyses were carried out using SPSS 19.0 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. All experimental data are shown as mean ± SEM. A probability of p ≤ 0.05 was considered statistically significant, and a probability of p ≤ 0.01 was considered to be extremely statistically significant.

 

Table I.- Primers of studied genes.

Gene Name	Primer sequence (5′→3′)	Length (bp)	Tm (°C)	Accession No.
BMPR1B	F: 5′-TGACGGACCTATACACCACA-3′ R: 5′-GTACCGAGGTCTGGCTTCTT-3′	121	60	NM_001142888.2
BMP15	F: 5′-TGTTGGGCAAAAGCTCTGGA-3′ R: 5′-GCCATGCCACCAGAACTCAA-3′	106	60	NM_001114767.1
GDF9	F: 5′-AACAGACGCCACCTCTACAA-3′ R: 5′-CACGATCCAGGTTAAACAGCA-3′	124	60	NM_001009431.1
Smad1	F: 5′-TGGTTCCAAGACACAGCGAATA-3′ R: 5′-GGTGTATCTGCTGGCATCTGAA-3	252	60	XM_015101506.1
Smad5	F: 5′-GCACAGCCTTCTGGTTCA-3′ R: 5′-GGGTAGGGACTATTTGGAG-3′	132	60	XM_012115987.1
Smad9	F: 5′-CCAGCACTCAGATTTTCGGC-3′ R: 5′-GCACTCGGCATAGACCTCTC-3′	147	60	XM_015098108.1
RPL-19	F: 5′-ATCGCCAATGCCAACTC-3′ R: 5′-CCTTTCGCTTACCTATACC-3′	154	60	XM_012186026.1

 

Results

Expression levels of BMPR1B, BMP15, GDF9, Smad1, Smad5, and Smad9

The expression levels of BMPR1B, BMP15, GDF9, Smad1, Smad5, and Smad9 in eight tissues (brain, cerebellum, hypothalamus, pituitary, testis, epididymis, vas deferens, and adrenal gland) in both high fecundity breed Small Tail Han sheep and low fecundity breed Sunite sheep were measured by qPCR in this study.

As shown in Figure 1, BMPR1B is expressed in all tissues with the highest level in the epididymis, followed by the hypothalamus, brain, and cerebellum. The expression of BMPR1B in the brain, hypothalamus, pituitary, epididymis, and adrenal gland is significantly higher in SNT than in STH (p < 0.01, p < 0.05).

 

As shown in Figure 2, BMP15 is specifically expressed in the epididymis and the expression of BMP15 in the epididymis is significantly higher in SNT than in STH (p < 0.01).

As shown in Figure 3, GDF9 is expressed in all tissues with the highest level in the brain, followed by the testis, cerebellum, and hypothalamus. The expression of GDF9 in the cerebellum and vas deferens is significantly higher in STH than in SNT (p < 0.05). The expression of GDF9 in the adrenal gland is significantly higher in SNT than in STH (p < 0.01).

As shown in Figure 4, Smad1 is expressed in all tissues with the highest level in the epididymis, followed by the cerebellum, brain, and hypothalamus. The expression of Smad1 in the brain and adrenal gland is significantly higher in STH than in SNT (p < 0.05). The expression of Smad1 in the vas deferens is significantly higher in SNT than in STH (p < 0.01).

 

 

 

 

As shown in Figure 5, Smad5 is expressed in all tissues with the highest level in cerebellum, followed by the brain, hypothalamus, and adrenal gland. The expression of Smad5 in the adrenal gland is significantly higher in STH than in SNT (p < 0.05).

As shown in Figure 6, Smad9 is expressed in all tissues with the highest level in the brain, followed by the cerebellum, epididymis, and hypothalamus. The expression of Smad9 in the brain and epididymis is significantly higher in SNT than in STH (p < 0.05, p < 0.01, respectively). The expression of Smad9 in the cerebellum and hypothalamus is significantly higher in STH than in SNT (p < 0.05).

 

Discussion

BMPR1B

BMPR1B (FecB gene) is one of the major fecundity genes in female reproduction; however, not much is known about the reproductive role of the BMPR1B gene in male reproduction. Previous reports have found that BMPR1B belongs to the type I receptors of BMPs (Aquino et al., 2017; Kaivo-oja et al., 2006), which figures prominently in the directional migration and proliferation of primordial germ cells (PGCs) (Dudley et al., 2007; Okamura et al., 2005) and precursor cells of sperm (Hammoud et al., 2014; Larriba et al., 2018). Therefore, the BMPR1B gene has a certain impact on male reproduction.

Studies have found that BMPR1B is widely expressed in the ovary, liver, hypothalamus, pituitary, uterus, heart, and muscle of mammals (Valdecantos et al., 2017; Goyal et al., 2017; Foroughinia et al., 2017). In ewes, BMPR1B is highly expressed in the reproductive tissues and moderately expressed in the brain, skeletal muscle, and kidney (Ciller et al., 2016; Tang et al., 2018; Wilson et al., 2001). In this research, BMPR1B was found to be expressed in all selected tissues and highly expressed in the brain, cerebellum, hypothalamus, and epididymis, which indicated that BMPR1B may have a role in both ewe and ram reproduction.

The expression of BMPRIB in the brain, hypothalamus, pituitary, epididymis, and adrenal gland is significantly higher in SNT than in STH. This observation was different from a previous study comparing prolific and non-prolific ewes (Xu et al., 2010), in which ewes with high fecundity were reported to have a higher expression of BMPR1B in the reproductive tissues, which implies that ram may have a different regulation mechanism in reproduction when compared to ewe. Considering the function of BMPR1B in the proliferation of PGCs, it seems plausible that BMPR1B may have a certain inhibitory effect on ram reproduction. Of course, further studies should be performed deeply to investigate the relationship between BMPR1B and ram reproduction.

BMP15

BMP15 is an important regulator of male germ stem cell (mGSC) proliferation and differentiation (Liu et al., 2018). Hu et al. (2017) reported that over-expression of BMP15 in goat mGSCs leads to the increased expression level of c-Kit, a gene that promotes spermatogonial differentiation and the proliferation of mGSCs. Thus, BMP15 is an important candidate gene in male fertility.

In 1998, Dube et al. (1998) explored the expression of BMP15 in several tissues, including ovary and testis in mice, and found that BMP15 is specifically expressed in the ovary. The expression of BMP15 in goats (Silva et al., 2005) and pigs (Li et al., 2008) is similar to that in mice. In contrast, Aaltonen et al. (1999) reported the expression of BMP15 in the testis and ovary in humans. Similarly, Pennetier et al. (2004) reported the expression of BMP15 in the testis and ovary in cattle. This study suggested that BMP15 is specifically expressed in the epididymis in rams. One potential explanation is that the differences in the genetic models led to these results.

It is known that BMP15 exerts its biological effects by initially interacting with a type II receptors of BMPs, which results in the activation and phosphorylation of BMPR1B (Moore et al., 2003). We compared the expression level of BMP15 and BMPR1B, the expression of BMP15 and BMPR1B in the epididymis in SNT was found to be significantly higher than in STH which implies that the expression level of BMP15 and BMPR1B may be negatively correlated with the fecundity of rams.

GDF9

For a long time, GDF9 was considered to be specifically expressed in the ovaries of animals, until Fitzpatrick et al. (1998) reported the expression of GDF9 in non-ovarian tissues including the testis, brain, pituitary and bone marrow. Earlier, the GDF9 expression was detected in the testis in rats, mice, humans, cattle (Tang et al., 2017), alpacas (Guo et al., 2013) and cats (Zhao et al., 2011). To our knowledge, no research on the expression of GDF9 in rams has ever been reported. In the present study, GDF9 was detected in all 8 tissues in rams, which implies that it plays a role in promoting the differentiation of many tissues. The highest expression of GDF9 in the epididymis further confirmed that GDF9 is associated with the epididymal function.

Because numerous studies revealed that GDF9 has promoting effects on genetic and cellular signaling levels and the mitosis of germ cells (Tang et al., 2013; He et al., 2012), we compared the expression level of GDF9 in the testis, epididymis, and vas deferens between two sheep breeds. We found no significant difference between the expression level of GDF9 in the testis and epididymis of SNT and STH, but the expression level of GDF9 in the vas deferens of STH is significantly higher than in SNT. Our findings are in agreement with Tang et al. (2017) who found exogenous GDF9 significantly promoted Sertoli cells (SCs) proliferation and inhibited the apoptosis of SCs which suggested GDF9 to have a supporting role for the maintenance of spermatogenesis. Therefore, we concluded that the prolificacy of ram might be due to the high expression level of the GDF9 gene.

Smad

Smad1/5/9 is widely expressed in mammals, especially in the brain and hypothalamus–pituitary–gonadal (HPG) axis (Wang et al., 2018a, b; Ohyama et al., 2015). The present study found that Smad1/5/9 is detectable in eight tissues of rams; meanwhile, the expression level of Smad1/5/9 in the brain, cerebellum, and HPG axis is higher than in other tissues, which is consistent with our expectation.

BMP type I receptors are transphosphorylated by type II receptors, resulting in cascades of Smad signaling (Aquino et al., 2017). Shi et al. (2016) found that the deletion of BMPR1B leads to an increased phosphorylation level and decreased expression level of Smad1/5/9 in male mice. To explore the expression pattern of BMPR1B in rams, we compared the expression level of BMPR1B and Smad1/5/9. We surprisingly found that the expression level of BMPR1B in the brain, epididymis, and adrenal gland is significantly higher in SNT than in STH; meanwhile, the expression level of Smad1/5 in the brain and adrenal gland was higher in STH than in SNT. The results show that BMPR1B signaling may be involved in some of those mechanisms in the brain and adrenal gland of rams; however, the expression level of Smad1 in the epididymis is significantly higher in SNT than in STH. We speculate that BMPR1B may have a certain inhibitory effect on the spermatogenesis of rams; however, the degree of the function of BMPR1B in rams remains to be investigated.

Studies have demonstrated that the BMP/Smad signaling pathway may be associated with the spermatogenesis process (Itman et al., 2010). Research on mice (Mendis et al., 2011) and rats (Chan et al., 2017) may provide an insight into the synergistic effect of BMP15 and Smad1/5/9 in male animals: the expression level of Smad1/5/9 is positively regulated by BMP signaling (Katakawa et al., 2016). Additionally, we compared the expression level of BMP15 and Smad1/5/9. The expression of BMP15 and Smad1 in the epididymis of SNT is significantly higher than in STH, but there is no significant difference between the expression of Smad5 and Smad9 in the epididymis of two sheep breeds. We provide evidence that the BMP/Smad signaling pathway may be associated with the spermatogenesis process in rams to some degree. Further research is necessary to draw conclusions.

 

Conclusions

In conclusion, we found that BMPR1B, GDF9, Smad1, Smad5, and Smad9 are expressed in all selected tissues and are highly expressed in the epididymis, whereas BMP15 is specifically expressed in the epididymis, which indicates that they may play important roles in the ovine epididymis and promote spermatogenesis. Our findings of ovine BMPR1B, BMP15, GDF9, Smad1, Smad5, and Smad9 will help to further understand their expression and function, and may contribute to exploring their role in the ram reproduction system. This is the first study of the six genes tissue expression pattern in rams.

 

Acknowledgements

This research was funded by National Natural Science Foundation of China (31772580, 31872333), Earmarked Fund for China Agriculture Research System (CARS-38), Agricultural Science and Technology Innovation Program of China (ASTIP-IAS13), China Agricultural Scientific Research Outstanding Talents and Their Innovative Teams Program, China High-level Talents Special Support Plan Scientific and Technological Innovation Leading Talents Program (W02020274), Tianjin Agricultural Science and Technology Achievements Transformation and Popularization Program (201704020), The Projects of Domesticated Animals Platform of the Ministry of Science, Key Research and Development Plan (modern agriculture) in Jiangsu Province (BE2018354), Major new varieties of agricultural projects in Jiangsu Province (PZCZ201739), Jiangsu Agricultural Science and Technology Innovation Fund (CX(18)2003), The Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Major projects of Natural Science Research of Colleges and Universities in Jiangsu Province (17KJA230001), The Project of six peak of talents of Jiangsu Province of China and Postgraduate Research and Practice Innovation Program of Jiangsu Province (SJCX18_0804).

 

Statement of conflicts of interest

All authors declare no conflicts of interest.

 

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