The RNAi vecter construction and verification of the functions in sexual control of Cervus elaphus Zfx gene

Background: Zinc finger protein X-linked ( Zfx ) was regarded to be a sex determination factor, and plays a critical role in spermatogenesis. RNAi is an effective method of silencing Zfx / Zfy mRNA expression. However, there has been little research on the use of RNAi technology to control the sex of the offspring of Cervus elaphus . The objective of this study was first to explore an efficient method to alter the Cervus elaphus offspring sex-ratio by silencing the genes Zfx during spermatogenesis. Results: Three recombinant expression vectors pLL3.7/A, pLL3.7/B and pLL3.7/C were constructed to interrupt the Zfx gene. The results showed that the expression of Zfx mRNA was significantly silenced by pLL3.7/A ( P < 0.01), compared with the control group. The group injected with pLL3.7/A produced 94 Cervus elaphus , including 68 males and 26 females. The male rates (72.34%) were significantly higher than the control groups ( P < 0.01). Conclusions: Our experiment suggest that the Zfx gene plays a significant role in the process of X-sperm formation. Zfx siRNA may be a useful approach to control offspring sex in Cervus elaphus .


Background
The Cervus elaphus is the second-class protected animal in China, and an important economic animal. It has high output of antler and great economic value. Antler is a kind of precious traditional Chinese medicine and a valuable nourishing health product [1,2]. Velvet antler is considered to have various pharmacological activity, such as immunomodulatory, wound-healing effects, anti-inflammatory and enhancement of sexual function [2][3][4]. The main purpose of raising deer is to obtain antler and only male produces antler, so the producers have great interest in obtaining more male fawns for the economic benefits of Cervus elaphus breeding. Therefore, in this study, RNAi technology was used to control the sex of the Cervus elaphus and improve the male ratio, which is an important means to increase the production of antler and increase economic benefits.
Up to now, flow cytometry (FCM) analysis has been the most accurate method for sperm separation, which accuracy can be reached up to 90% [5]. But it is relatively inefficient, requiring specialized, expensive and immobile equipment as well as skilled technicians. Therefore, an alternative way is to find a new sex control method which could interfere with X chromosome generation during spermatogenesis. Zfx/Zfy is thought to play a major role in sex determination during spermatogenesis, which is located on the X/Y chromosome [6][7][8][9]. The binding of zinc finger protein motifs to nucleic acid binding proteins plays an important role in sex determination of early embryos [10,11]. Recent reports on mice and humans have shown that Zfy is essential for sperm formation, fertilization and reproduction [12].
Zfx is concerned with X spermatogenesis, can serve as a strong transcription activation factor, and guides the target genes positioning in X sperm nuclei through the nuclear membrane, it may also have a transcription activation function for some genes during X spermatogenesis [13]. Silencing the expression of Zfx may interfere with the normal function of these sex-specific genes after meiosis I [14]. Therefore, simple methods of identifying Zfx at the level of RNA alone may allow for directional manipulation of X or Y sperm, respectively.
The RNAi technology has the advantages of simple operation, low cost, quick effect, high accuracy and good repeatability. RNAi is an effective method of silencing Zfx/Zfy mRNA expression [14][15][16][17]. However, there has been little research on the use of RNAi technology to control the sex of the offspring of Cervus elaphus. Therefore, silencing Zfx expression in testis by RNAi during spermatogenesis may change the sex ratio of Cervus elaphus offspring. In this study, specific RNA interference fragments were first designed to interfere with development of X sperm, in order to change the male proportion of Cervus elaphus offspring and improve the market economic benefits of Cervus elaphus industry in world.

Spermatogenic cells separation and transfection
Collagenase IV, hyaluronidase and trypsin were used to digest the testicular tissue of Cervus elaphus into spermatogenic cells and Sertoli cells, the segregated spermatogenic cells were round and full in shape with a clear outline and uniform size. However, the isolated Sertoli cells were irregular, mostly of them were polygonal and triangular (Fig. 1). After culturing 24 h, reconstructed vectors were transfected into cultured cells. There was no fluorescence expression at 24 h after transfection, however, there was some fluorescence expression at 48 h, and the highest fluorescence expression was at 58 h, moreover the transfection efficiency could reach about 92%. However, at 72 h, the viability of the cells decreased, and some cells showed apoptosis. So, after transfection for 58 h, the cells mRNA was extracted and then reverse-transcribed into cDNA. The results of transfection at 58 h showed that the Zfx gene RNAi vector were successfully transferred into spermatagonia of the Cervus elaphus (Fig. 2).
The expression of Zfx mRNA in injection groups and control groups by qRTPCR The Zfx mRNA levels results showed that all recombination vectors were lower than the control groups which were the blank and vector-free group (Fig. 3). Specifically, the Zfx mRNA level of test groups injected with pLL3.7/A (P < 0.01) were significantly lower than control groups, and transfected pLL3.7/B were lesser than the control group (P < 0.05). Therefore, pLL3.7/A vector was selected for use in the in vivo experiment because it had the least expression of Zfx mRNA (P < 0.01) ( Table 2). Table 2 Zfx RNAi oligonucleotide sequence with HpaI and XhoI enzyme sites.

Vector shRNA
Oligonucleotide sequence pLL3.7 A F: Notes: Using pLL3.7 as the vector construction of shRNA interference fragment.

Statistical analysis sex proportion of the offspring
The non-injection group, as control group, produced 98 Cervus elaphus, including 50 males and 48 females. While, the group injected with pLL3.7/A produced 94 Cervus elaphus, including 68 males and 26 females. The male rates (72.34%) were significantly higher than the control groups (P < 0.01) ( Table 4). In addition, the fertility rate, dystocia rate, abortion rate, reproduction rate, empty embryo rate, offspring survival rate and offspring deformity rate of the female deer experimental group were not abnormal compared with the control group. This result suggest that the Zfx gene plays an important role in the process of Xsperm formation. Zfx siRNA is an advanced method to control Cervus elaphus X spermatogenesis and sex ratio of offspring.

Discussion
Although the reproduction of sex determination mechanism requires multiple gene interactions, the sex ratio in nature is stable at 1:1 [18]. The formation of X or Y sperm during spermatogenesis plays an important role in sex determination of offspring [19]. Previous studies in our laboratory found that Zfx or Zfy siRNA can change the sex ratio of animal offspring, such as cows, mouse, sheep, swine [14,17,20]. Cervus elaphus is a special economic animal and its antler has great market economic value. However, there has been little research on the use of RNAi technology to control the sex of the offspring of Cervus elaphus. In our study, the Cervus elaphus Zfx siRNA was first designed to change the sex ratio of the offspring, once again demonstrating the important role of Zfx in gender. More importantly, it is of great significance to increase the male ratio of Cervus elaphus offspring to increase antler yield and economic benefits.
At present, there are only a little research on Zfx gene silencing to make the sex shift of offspring, so the mechanism of action of Zfx in sperm is not clear. Some studies have shown that reducing the level of Zfx/Zfy mRNA expression will make the physiological function of X/Y sperm worse, which can influence offspring gender [14,17,[20][21][22]. Zfy is closely related to sperm head and tail formation and neck development [23], which is highly expressed between meiosis I and II. It can regulate sperm morphology and ROSI efficiency, and promotes second meiosis [24,25]. Similarly, Zfx gene is female-specific after meiosis I and is always expressed throughout spermatogenesis [13]. The high expression of Zfx in round sperm cells of adult mouse testis is closely related to the self-renewal of spermatocytes. Generally speaking, Zfy gene is to ensure the occurrence of the second meiosis, and the Xrelated gene Zfx on the chromosome has a promoting effect [26]. Reducing the expression level of Zfx mRNA will affect the development of haploid X sperm, which will lead to the decrease of female offspring [14].
However, there was no health abnormality and decreased reproductive performance Therefore, silencing of the Zfx maybe only interferes with the formation of haploid X sperm, but not the normal development of embryo.
Spermatogenesis is a highly regulated and orderly process. It is regulated by many genes, but its mechanism is still unclear [28,29] and further research is needed.
However, this study, the Zfx gene silencing technology was successfully used to significantly increase the number of male Cervus elaphus in the offspring, which not only increased the economic benefits in production, but also provided theoretical reference for the subsequent research on gender control mechanism in scientific research.

Conclusion
The results of this study indicate that the use of RNAi to interrupt Zfx may be a useful approach to control Cervus elaphus sex which lays a foundation for producing sex control semen.

Experimental animals
Healthy Cervus elaphus were selected from the Xinjiang Production and Construction

Construction of PLL3.7-shZfx recombination vectors
Zfx siRNA sequences were evaluated and selected by Cervus elaphus Zfx sequence (KP257294.1) and siRNA design principles [30,31]. According to the design principle on siRNA, preliminary screening of the designed siRNA sequences was processed, and then the siRNA sequences were selected and compared the homology with Cervus elaphus Zfy sequence (MN560153) through BLAST. Nonspecific fragments were eliminated and 3 specific siRNA sequences was finally selected (Table 1)  Injection(100 mg/ml) to make it lose consciousness, then the common artery was cut to bleed, and it died immediately. Then take the testicles aseptically, put them in the ice box, and reach the laboratory within 1 h. The improved two-step enzymatic digestion method was used to isolate spermatogenic cells of the Cervus elaphus testis [32].

The RNAi efficiency of each vector in vitro
The mRNA level of Zfx and Gapdh (Table 3)

Consent for publication
Not Applicable.

Availability of data and material
Cervus elaphus Zfx sequence and Cervus elaphus Zfy sequence are respectively archived at NCBI GenBank under accession numbers KP257294.1 and MN560153.

Competing interests
The authors declare that they have no competing interests.

Funding
The funding was provided by High-level talent research start-up fund project supporting XinJiang from Production and Construction Corps of ShiHeZi University, China (RCZK2018C13). The funding agency had no input to the design of the study or in the collection, analysis or interpretation of data.