Various gene modification techniques to discover molecular targets for nonhormonal male contraceptives: A review

Abstract The identification and characterization of relevant targets are necessary for developing nonhormonal male contraceptives. The molecules must demonstrate that they are necessary for reproduction. As a result, a sophisticated technique is required to identify the molecular targets for nonhormonal male contraceptives. Genetic modification (GM) techniques are one method that can be applied. This technique has been widely used to study gene function that effected male fertility and has resulted in the discovery of numerous nonhormonal male contraceptive target molecules. We examined GM techniques and approaches used to investigate genes involved in male fertility as potential targets for nonhormonal contraceptives. The discovery of nonhormonal contraceptive candidate molecules was increased by using GM techniques, especially the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 method. The discovery of candidate nonhormonal contraceptive molecules can be a wide-open research for the development of nonhormonal male contraceptives. Therefore, we are believing that one day nonhormonal male contraceptives will be released.


Introduction
Until now, women dominated contraceptive methods, and men were under-represented (1,2). Despite varying contraceptive methods, unplanned pregnancy rates remained high (3)(4)(5). Male contraceptives include condoms and vasectomy. Contrary to popular belief, vasectomy is irreversible and less reliable than female contraception (4). Hormonal male contraceptives work by interrupting the negative feedback loop of hypothalamus-pituitary-gonad axis.
This method of contraception should be revisited due to several problems and negative effects. Nonhormonal male contraceptives have been created to avoid male hormonal contraceptive side effects (6). One of the candidates for nonhormonal male contraceptives is the epididymal molecule which is suspected to play a role in sperm maturation (7)(8)(9)(10). The development of nonhormonal male contraceptives by targeting proteins or genes involved in male fertility is promising for controlling male fertility. A consistent strategy for investigating gene function is necessary to identify candidate genes for use as biomarkers or contraceptive targets. The best way to determine gene function is by genetic modification (GM) techniques in animal model. Common GM approaches include transgenic, knockdown, and knockout/knockin/gene-trapped (11,12).
More than 400 genes that play a role in male fertility have been analyzed using animal models (11). This number is less because thousands of genes play a role in the regulation of the complex process in male fertility. GM techniques are developing rapidly. This was followed by an increasingly fast and easy way to make animal models which resulted in more findings of genes that play a role in male fertility as candidates nonhormonal male contraceptives. Although there are many candidate molecular targets for nonhormonal male contraceptives, the development and progress of nonhormonal male contraceptives are slow. The scope of the problem in this article is GM techniques for producing animal models, various genes that play a role in male fertility as nonhormonal male contraceptive candidates, and the development of nonhormonal male contraceptives with reported molecular targets.

Material and Methods
The research for relevant articles to this review was acquired from search engines particularly Google Scholar, PubMed, Elsevier, and Scopus distributed between January 2003-December 2021. The keywords used were contraceptive, nonhormonal, GM techniques, animal model, and molecule candidate of male contraceptive. The articles were observed and specified for the current review. This study concentrates on discovering molecule candidates of nonhormonal male contraception from various GM techniques. We categorize 13 articles about the generation of making animal models for infertility by using transgenic, knockout, or Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) techniques. A total of 27 articles in which researchers examined genetically modified to produce sterile or decreased fertility in males were found. Genes that affect infertility in males but do not affect hormones were used as candidates for nonhormonal male contraceptives. Furthermore, a search for candidate nonhormonal male contraceptive molecules was conducted and 26 articles were collected.

Animal model using GM
More than thousand genes are involved in spermatogenesis. As spermatogenesis is complex and there are multiple causes of aberrant spermatogenesis, using experimental animal models with genetic alteration techniques is particularly advantageous. Mice are the most often used experimental animals because they have a short reproductive cycle, genetics similar to humans, and mouse embryos are easily genetically modified (12). Using GM techniques to create experimental animals allows genes from one individual to be transferred to another or between species.

Transgenic
Transgenics are genetic alterations generated through recombinant DNA insertion that can be passed down from generation to generation. Endogenous or exogenous genes or DNA fragments are introduced to boost expression (11). Initially, transgenic individuals were generated through nuclear transfer and microinjection into the pronucleus. Microinjection has persisted because it is more stable than nuclear transfer (12). The advantages are that almost all cells from these transgenic offspring contain transgenes, production is relatively quick, and the effects of gene overexpression can be analyzed and studied to determine the gene's utility. While disadvantages are integrated randomly in a small percentage of cases, there is a risk of DNA entering critical loci, resulting in genetic mutations, nonphysiological phenotypic effects. Transgenes can enter gene loci silencing (13,14).

Knockout
The term "knockout" refers to animals whose target genes have been deleted by deleting critical exons to prevent transcription. One strategy for deactivating a gene is to delete its critical exon and replace it with an antibiotic-resistant gene, such as the neomycin gene (neo ). After electroporation of the constructed gene into embryonic stem cells (ESCs), the cells are grown in a medium containing the antibiotic neomycin.
As a result, the transgenic ESCs will survive in a neomycin-treated medium. Additionally, ESCs containing recombinant DNA will be injected into the blastocyst to generate chimeras. Then, chimeric organisms are mated with wild-type organisms to generate heterozygous offspring.
This heterozygous offspring will be crossed with another heterozygous offspring to generate homozygous mutant/knockout offspring (12).  ESCs was isolated and cultured from the inner cell mass of wild-type mice blastocysts. Selected ES cells were injected into blastocysts from the other strains and implanted into pseudopregnant female mice. Chimeric progeny will be produced. To generate heterozygous knockout offspring, chimeric offspring were mated with wild-type mice. Heterozygous offspring can be mated to generate homozygous knockout mice.
Despite knockout widespread use, this technique is still time-consuming and expensive than transgenic techniques. Knockout can result lethal embryonic or neonatal death caused by target gene is expressed at early embryonic or many tissues that are important for support in neonatal. So they cannot be evaluated postnatally or in adulthood. Another disadvantage of standard knockout is the difficulty distinguishing direct effects on cells or tissues from secondary effects on other organs caused by indirect actions. As a result, it is critical to keep in mind that the observed phenotype may result from both direct and indirect actions of gene products (12,15). The solution to these problems is to develop knockout techniques such as conditional knockout and knockout inducer. Conditional knockout occurs in specific tissues and organs using the cloning vector pAW8-yEGFP (Cre)-locus of X over (lox) system. The Cre-Lox system comprises 2 components: the Cre recombinase and the recognition site loxP (16).
2 mutant animal strains must be used to generate mutants using the Cre-loxP system. The Cre strain was expressed in a specific tissue or organ in the first animal. The second animal is a strain of animal that has a loxP recognition site flanking the target gene that will be excised/cut in a unidirectional orientation. Additionally, the offspring of these 2 animal strains were mated to ensure that target genes were knocked out only in specific tissues/organs. This occurs because Cre is expressed in a limited number of tissues/organs. The Cre protein recognizes the loxP recognition area between the target gene, resulting in the target gene's excision and truncation (17). Procedure conditional knockout can be seen in figure 3. . The Cre-loxP system is used to generate conditional knockout experimental animals. Animals with lox in the target gene and animals with Cre expressed exclusively in certain organs/tissues. If Cre is expressed, the offspring will be knockout; if Cre is not expressed, the offspring will be wild type.
The advantages of conditional knockout are flexibility of the Cre-loxP system, can assess the role of target genes at various developmental stages, and enables analysis of the target genes role at a variety of sites. The disadvantage of this method is that it is difficult to identify a promoter that will specifically direct Cre expression to the desired tissue (16,17).

Clustered regularly interspaced short palindromic repeats (CRISPR)
CRISPR is a type of DNA segment found in prokaryotes (bacteria and archaea) consisting of sequences or sequences of nucleotides with short repetitions. The CRISPR gene functions as an immune system in these prokaryotes, protecting them from infection, conjugation, and transformation caused by foreign genetic material (14). CRISPR-Cas9 can be used to edit genes depending on the system's capability. In contrast to transgenic techniques, which perform genetic engineering on a genome randomly, the CRISPR-Cas9 system performs targeted and precise genetic engineering ( Figure 4) (20).
The primary benefit of this technology is its ability to automate complex gene targeting vector designs and ESCs manipulation, thereby reducing modeling time and costs. Additionally, CRISPR/Cas9 is not restricted to a rodent's particular strain or genetic background, as ESCs is not required (21). CRISPRbased gene editing makes knockout easier.
One disadvantage of the CRISPR/Cas9 system is that it is inefficient at inserting large DNA sequences (22). To overcome this limitation, it was reported in 2017 that the addition of a CRISPR single stranded DNA (ssDNA) insert (Easi-CRISPR) could be used.
These donor sequences can range in length from 500 nt to 2 kb and have been shown to provide highly efficient targeted insertion (23). The advantage of Easi-CRISPR is that long donor ssDNA sequences do not randomly integrate into the genome, as is the case with short donor dsDNA sequences. Additionally, this donor serves as a superior template for homology repair compared to conventional CRISPR sequences. While Easi-CRISPR appears to be a significant advancement over basic CRISPR technology, ensuring the quality and precision of extremely long ssDNA sequences. The resulting RNA will be reverse-transcribed using reverse transcriptase to generate this sequence.
Unlike DNA-dependent polymerases, this enzyme is prone to errors and cannot correct them. This results in sequences that may contain mutations (23).

Male infertility genes as a nonhormonal contraceptive candidate
The testes and epididymis are 2 organs that are involved in the process of male infertility. The testes are involved in spermatogenesis, while the epididymis is involved in sperm maturation. As a result, numerous studies have examined male infertility at the genomic, transcriptomic, and proteomic levels in these 2 organs. Along with organ analysis, molecular analysis at the cellular level, specifically spermatozoa, was performed. Obtaining a contraceptive target molecule is one of the objectives of the molecular study of male infertility.
The number of genes and proteins thought to play a role in male fertility must be established by understanding their role and function in fertility.
GM techniques, particularly knockout, are one method for analyzing the function and role of genes. Fertility analysis must be performed in vivo due to the complexity of the reproductive process. As a result, it is essential to analyze the function and role of male fertility genes. In table I, we list the genes involved in fertility that have been characterized using knockout animal models.

Brca2
Spermatocytes stop early in prophase I 26

Crisp2/Crisp4
Inflammation of the epididymis resulting in decreased sperm viability and decreased fertility rates 27

Catsper and KSper
Infertility without apparent systemic effects 28

Ercc1
Increased DNA damage in testes, increased apoptosis in germ cells 29

Eif4g3
Stopped at the stage of meiosis, spermatocytes fail to exit prophase through the G2/MI transition 30

Eppin
Reduction of sperm motility 31

Gpx4
Sperm count decreased significantly, infertility in males, reduced sperm motility, swollen mitochondrial membrane 32

Fkbp6
Stopping at the pachytene stage and increasing apoptosis in germ cells 33

Tmem95
Completely infertile or severely subfertile 45 Spermatozoa become fertile during their transit through the epididymis. Microarray analysis identified over 17,000 genes expressed in the epididymis, but only a few are expressed in the epididymis to a limited extent. To investigate the function of highly expressed genes in the epididymis in vivo, experimental animals in the form of mice were created that were deficient in 9 genes found to be highly expressed in the head and corpus epididymis (Pate1, Pate2, Pate3, Clpsl2, Epp13, Rnase13, Gm1110, Glb1l2, and Glb1l3). The CRISPR/Cas9 system was used to generate knockout mice. The epididymis histology and sperm morphology of all knockout lines were identical to those of control males. Females of wild-type mated with knockout males produced the same number of offspring as control males. Thus, 9 genes that were abundantly expressed in the head and corpus epididymis were discovered to be secreted for sperm function and male fertility. The generation of knockout mice using CRISPR/Cas9 accelerates screening genes expressed in the epididymis for potential reproductive functions (48). According to animal models, the target may be effective if specificity is increased to limit off-target effects.

Development of nonhormonal male contraceptives
The first candidate molecule for nonhormonal male contraceptives is retinoic acid. It was discovered that male sterility could be caused by vitamin A deficiency (49). Table II showed details of the development of nonhormonal male contraception.  (58) It is unclear whether this approach would be more suitable for female use because of the impact on progesterone function in the fallopian tubes (58)

CDB-4022
Mitogenactivated protein kinase (MAPK) pathway activation in Sertoli-germ cell junction Inhibits the mature sperm (61) Reversible decrease in sperm production with no apparent side effects (61)

6.
Testis-specific serine/threonine kinases (TSSK) Spermatogenesis and sperm function (53) GSK2163632 A TSSK2 inhibitor Loss of fertility in mice (53) It is necessary to identify selective molecules in inhibiting TSSK2 because this compound can inhibit other kinases than TSSK2.
Inconsistent side effects was leaded by the nonselective action of this molecule (53)

CatSper
Capacitation, hyperactivation of motility, and the acrosome reaction (54) Nifedipine Calcium channel blocker (54) Epididymal sperm counts, motility, and fertility of male BALB/c mice significantly decreased (54)  Reduced fertilization and embryo development in the highest concentration of CK-636, but after treatment, fertilization significantly increased, whereas embryonic development significantly decreased (65)

S-Allylcysteine (sAC) inhibitors
Decreased male fertility through reduced sperm motility and capacitation (70) The correlation between dominant absorptive hypercalciuria and ADCY10 has not slowed the development and optimization of ADCY inhibitors as nonhormonal contraceptives (70

Discussion
Proteomic

Conclusion
A good contraceptive must meet a number of criteria, including being safe, reliable, effective, affordable, easy to use, without serious side effects, readily available, and reversible. It are optimistic that one day nonhormonal male contraceptives will be developed and it will be effective and beneficial to the wider community.