Unlocking Genetic Mysteries during the Epic Sperm Journey toward Fertilization: Further Expanding Cre Mouse Lines

Abstract

The spatiotemporal expression patterns of genes are crucial for maintaining normal physiological functions in animals. Conditional gene knockout using the cyclization recombination enzyme (Cre)/locus of crossover of P1 (Cre/LoxP) strategy has been extensively employed for functional assays at specific tissue or developmental stages. This approach aids in uncovering the associations between phenotypes and gene regulation while minimizing interference among distinct tissues. Various Cre-engineered mouse models have been utilized in the male reproductive system, including Dppa3-MERCre for primordial germ cells, Ddx4-Cre and Stra8-Cre for spermatogonia, Prm1-Cre and Acrv1-iCre for haploid spermatids, Cyp17a1-iCre for the Leydig cell, Sox9-Cre for the Sertoli cell, and Lcn5/8/9-Cre for differentiated segments of the epididymis. Notably, the specificity and functioning stage of Cre recombinases vary, and the efficiency of recombination driven by Cre depends on endogenous promoters with different sequences as well as the constructed Cre vectors, even when controlled by an identical promoter. Cre mouse models generated via traditional recombination or CRISPR/Cas9 also exhibit distinct knockout properties. This review focuses on Cre-engineered mouse models applied to the male reproductive system, including Cre-targeting strategies, mouse model screening, and practical challenges encountered, particularly with novel mouse strains over the past decade. It aims to provide valuable references for studies conducted on the male reproductive system.

1. Introduction

Conditional knockout (cKO) with recombinant enzymes allows for the removal of targeted genes to investigate their functions in physiological processes. Recombinase systems, including the cyclization recombination enzyme/locus of crossover of P1 (Cre/LoxP), vCre/vLoxP, sCre/sLoxP, Flp/FRT, Dre/Rox, and Vika/Vox [1,2,3,4,5], are utilized with Cre/LoxP being the most prevalent. Cre, a 38-kDa recombinase with site specificity, has been an in vivo molecular tool for over 30 years [6,7]. It recognizes DNA sequences between two LoxP sites (Figure 1A). The target DNA sequences flanked by LoxP, engineered artificially, are termed “floxed” and can be specifically ablated by Cre driven by the promoters of cell-specific genes after recombining downstream of the promoters via transgenic technology or knock-in (KI) into endogenous genic loci [8,9]. However, in most cases, the promoter in Cre-constructed vectors is incomplete and lacks distal regulatory elements (e.g., enhancers), which further reduces transcription efficiency (Figure 1B). In addition, the randomness of genomic insertions can result in uncontrollable recombination sites [10] and may exert toxic effects when multiple copies are expressed in cells [9]. Notably, as the homozygous ectopic expression of Cre lines behaves more unpredictably, the heterozygous Cre-engineered mouse was generally crossed with the flox-transgenic mouse, significantly decreasing breeding efficiency. Furthermore, the KI approach facilitates gene insertion into well-defined genic loci [11]. The CRISPR/Cas9 system, a DNA targeting and editing approach, has been broadly employed to establish Cre recombination mouse models driven in situ by target promoters after site-directed Cre insertion [12]. This method requires less time and is less costly for generating the desired transgenic mouse lines [13]. Functional gene investigation employing Cre transgenic mouse strains generated by CRISPR/Cas9 is more reliable and repeatable because the insertion locus is clear, promising endogenous gene expression away from interference to a large extent [13] (Figure 1B).

Spermatogenesis, which occurs in seminiferous tubules as the foundation of male fertility, is a complex process that produces mature gametes. It involves spermatogonia proliferation and differentiation into spermatocytes, spermatid generation through meiosis, and spermatozoa release into the tubule lumen after spermiogenesis [14]. Any aberration in these processes may impede fertilization. A systematic and in-depth investigation of the mechanisms regulating the orchestrated stages of spermatogenesis is crucial for treating male infertility or, conversely, the design of male contraceptives that target the desired developmental stages. Cre/LoxP recombination has been extensively utilized in specific DNA modifications targeting spermatogenic cells, such as Ddx4-Cre [15] for spermatogonia, Sycp1-Cre [16] for spermatocytes, and Prm1-Cre [17] for spermatids. Hammond et al. (2009) [18] and Smith et al. (2011) [19] reviewed genetic tools, including Cre recombinases specific for male and female germ cells and markers used to characterize cellular behavior or purify living germ cells. However, many subsequent reports with conditional ablations using these Cre have shown that they are not as specific or efficient as previously thought. Issues such as the efficiency and specificity of Cre recombinase not aligning with presuppositions need to be addressed given the widespread availability of abundant Cre-engineered lines, including heterotopic expression of non-target tissues [20,21], leakage [22], the instability of KO efficiency [17,23], and global KO [15,24]. Furthermore, various novel Cre mouse lines have been engineered with the request for more detailed dissection in critical cellular processes during spermatogenesis over the past decade (Figure 2). In this review, we summarize the novel advances in these events, especially the removal of targets in the primordial germ cells (PGCs), testis, epididymis, prostate, and seminiferous duct, as well as the unintended consequences of crossing with Cre mouse lines to provide a referential basis for studies investigating the functional roles of genes in male reproduction.

2. Cre Lines for PGCs and Spermatogonia

2.1. Ddx4-Cre

Ddx4, also known as Vasa or Mvh, is located on mouse chromosome 13 and is an ATP-dependent RNA helicase that is highly conserved in mammals, belonging to the DEAD (Asp-Glu-Ala-Asp) box family [25]. Ddx4 is primarily detected in germ cells with lower levels in other tissues such as the thymus and pancreas [26], appearing as early as embryonic day 10.5–12.5 (E10.5–12.5) and persisting in both the testes and ovaries throughout life [27,28]. Male infertility ensues after Ddx4 KO [27], while the Ddx4 mutation has minimal influence on female fertility, although oocytes with elevated levels of Ddx4 have been observed [29]. Ddx4-Cre lines (Strain #:006954) are available for functional analysis, particularly concerning genes implicated in spermatogonial establishment, maintenance, and differentiation. Notably, Ddx4-Cre-mediated target recombination efficiently initiates at E15, as indicated by the Rosa26-lacZ reporter, but it was inconsistent with its endogenous expression [30]. Recently, an improved Cre (iCre) line targeted by CRISPR/Cas9 exhibited higher efficiency in PGCs, with Cre activity initiated as early as E10.5. Furthermore, the probability of descendants with global KO from paternal Cre carriers was lower than that of Ddx4-Cre lines [30] for special floxed alleles, such as Cripto^flox [15]. However, both types of Cre carriers in females can lead to offspring with global KO, demonstrating a robust maternal effect [15,30]. Moreover, reports on the minimal ectopic activity of Ddx4-Cre/iCre recombinase have surfaced [15,30].

CreER constructs have been used for temporal gene ablation, with Cre fused to the mutated human estrogen receptor (ER) and named CreER [31]. CreER^T, as a human ER variant, was more sensitive to 4-hydroxytamoxifen (4-OHT), the metabolite of tamoxifen (TAM), compared to endogenous 17β-estradiol [32,33]. The mutants of CreER^T1 and CreER^T2 display even higher sensitivity to 4-OHT [34]. Moreover, CreER^T2 has been used extensively due to its robust activation at a specific stage upon TAM treatment with minimal background in Cre activity [34]. Given the unknown insertion site of Ddx4-CreER^T2 acquired through random gene recombination, Hoai et al. established a novel bicistronic mouse strain via CRISPR/Cas9, known as B6-Ddx4^{em1(CreERT2)Utr} [20]. However, infertility was found in the homozygous mouse line, manifesting as spermiogenesis arrest and absent mature spermatozoa. The ATPase activity of Ddx4 was halved in homozygotes, possibly due to the additional 25 amino acid residues introduced by the P2A sequence, which protects Ddx4 from being knocked out. Additionally, Cre recombination is primarily activated in the testes and ovaries but not in the pancreas or thymus [20]. In summary, novel-inducible B6-Ddx4^{em1(CreERT2)Utr} mice are available for studying sterility at specific spermatogenic stages, and the use of the heterozygote Cre line during mating is recommended.

2.2. Stra8-Cre

Stimulated by retinoic acid 8 (Stra8), localized on mouse chromosome 6, it is specifically expressed in germ cells and is essential for initiating meiosis during spermatogenesis and oogenesis, the deficiency of which leads to abnormal chromosomal behavior [35]. In females, Stra8 expression coincides with oocyte meiosis initiation at E13, with the peak at E14.5, and declines sharply after E16.5 [36]. In males, Stra8 expression is detected at 5 days post-parturition (DPP), with reports also indicating expression at 3 DPP, persisting in preleptotene spermatocytes [37]. As early as 2008, the Stra8-Cre line (Strain #:008208) was generated to study male germ cell development with a recombination efficiency of >95%, whereas the Cre line did not work in transgenic female mice [38]. The diverse strategies of mating with Stra8-Cre resulted in different efficiencies and offspring phenotypes. Bao et al. constructed an iCre regulated by the Stra8 promoter, excising the Mov10l1 flox allele with high efficiency in spermatogenetic cells. However, it failed to remove two Mov10l1 flox alleles in single breeding, and the testicular phenotypes were less severe compared to completely knocked-out mice [39]. Additionally, Balnco et al. observed the presence of the DOLT1 protein in offspring born to mice with a single allele of Dotl1 excised by the Stra8-Cre recombinase line [40]. This discrepancy could stem from the transgenic mice driving Cre expression using only part of the Stra8 promoter, which differs from the endogenous Stra8 expression profile. The uncertain insertion site and varying copies of Cre may also result in lower KO efficiency. Recently, the functional responsive elements in the 2.9 kb promoter lying upstream of 1.4 kb promoters that promised optimal Stra8 expression in vivo were characterized [41]. Therefore, Cre inserted into the targeted locus of Stra8 may exhibit optimal recombinase activity. The CRISPR/Cas9-mediated targeted mutation was employed to insert Cre or EGFP-Cre at the stop codon of Stra8 to study specific genes involved in spermatogenesis [42]. Avery et al. knocked in engineered P2A-Cre via the CRISPR/Cas9 system and recombinase activity without disturbing Stra8 expression or mouse fertility. It is worth noting that the Stra8-P2ACre line also exhibited Cre activity in female mouse germ cells at E12.5–E16.5 [43]. Xue et al. generated germline-specific Nat10 cKO females using Stra8-GFPCre or Zp3-Cre KI and revealed Nat10-indispensable oocyte meiosis progression, growth, and maturation [44]. The Stra8-Cre female strain constructed by targeted insertion contributes to studies focusing on oogenesis; however, offspring suffering from global KO appeared upon mating with the female Stra8-Cre line because of the long half-life of Stra8-Cre expressed in the oocyte.

2.3. PrP-CreER^T

Prp is primarily localized in the central nervous system (CNS) and shows low expression in the spleen, kidney, heart, and testes [45]. Specific PrP fragments or reporter genes were engineered for expression in the CNS. Philipp et al. reported that one of the transgenic lines expressing CreER^T induced by TAM and driven by 7.5 kb PrP fragments was restricted to the testis, with recombination occurring in particular in the spermatogonia, spermatocytes, and spermatids [46]. Following treatment with TAM for 6 days, approximately 30% of the floxed alleles underwent recombination in the testis without ectopic expression in other tissues, and even after 6 weeks of induction, 50% of seminiferous tubules still showed Cre activity [46]. Despite lower recombinant activity, this mouse strain provides a relatively specific tool for the genetic analysis of germ cells.

2.4. Dppa3-MERCre

Dppa3 (also named Stella or Pgc7) marks primordial germ cells emerging at E7.25, with robust expression persisting at around E15.5 in males and E13.5 in females [47,48]. Dppa3 is also expressed from the zygotic stage onward and remains in all blastocyst cells at E4.5 [48,49]. A novel transgenic mouse line (Dppa3-MERCre) was generated by Takayuki et al., and Cre recombinase expression was regulated by the Dppa3 promoter under simultaneous induction with 4-OHT, binding to domains flanking the Cre sequence [50]. Additionally, these strains demonstrated specific and efficient KO outcomes in PGC development, preimplantation embryos, and oocyte growth after 4-OHT stimulation [50]. Thus, Dppa3-MERCre can serve as a strain for exploring gene function during germ cell lineage development. Inducible KO also contributes to defining the function of specific genes causing embryonic lethality after target silencing. However, determining the optimal dose of the inductive agent before treatment in pregnant models is crucial to avoid miscarriage or other toxic effects associated with the drug.

2.5. Eomes-CreER^T

Eomesodermin (EOMES) is a T-box transcription factor crucial for intrinsic functions in immune cell development [51]. In addition, Eomes is vital for gastrulation formation and trophoblast development. Mutation in Eomes blocks at the blastocyst phase, preventing differentiation into trophoblasts after Eomes inactivation in the trophectoderm, suggesting its potential role in trophoblast stem cell physiology [52]. Stefanie et al. established an inducible CreER^T that carried 4-OHT in the MEFs sequence at the Eomes locus (Eomes-CreER^T) to study Emoes expression in pluripotency reprogramming in adult somatic mammalian cells [53]. Moreover, Eomes is localized in a subset of undifferentiated spermatogonia and contributes to regeneration after chemical injury and spermatogenesis regulation [54]. This suggests its potential to uncover target functions by establishing CreER^T recombination in germ cell lines via in situ TAM induction.

2.6. Ngn3-Cre

NGN3 is a marker of postnatal undifferentiated spermatogonial cells from 3 DPP [55] and is localized in the developing CNS, adult enteroendocrine cells, and pancreas [56]. Two Ngn3-Cre mouse strains were established for functional research on SSCs and spermatogenesis. Originally, Cre recombinase was constructed on an artificial bacterial chromosome (BAC) and inserted into the mouse Ngn3 locus 23 kb region [57]. A strong recombination signal was observed in the seminiferous tubules at 7 DPP, corresponding to the start of endogenous Ngn3 expression [57], with an efficiency close to 100% observed in juvenile and adult testes [57,58,59]. Another Ngn3-Cre mouse line (Strain #:005667) was generated by inserting a Cre recombinase cassette next to the start codon of the endogenous Ngn3 [60], with Cre catalytic activity appearing at 7 DPP [61,62] (Figure 2). Studies focusing on the action of SSCs [63,64] and germline development [65] during spermatogenesis have been conducted using this transgenic strain. Given the multiple localizations of NGN3 in the pancreas, CNS, and enteroendocrine cells, spatial specificity is gaining increasing attention.

2.7. Other Cre Lines for PGCs and Spermatogonia

Other Cre lines available for functional studies of PGCs and spermatogonial targets are described in Table 1. Tnap-Cre in PGCs was specifically designed to excise targets at E9.5–E10.5, while broad expression patterns were detected in the neural tube, placenta, labyrinthine region, and intestine after mid-gestation, achieving an efficiency of approximately 50% [66]. Nanos3 localizes to migrating PGCs and stages after settling in the gonads of both males and females. Nanos3-Cre bioactivity was observed at E7.75, with an efficiency ranging from 11 to 25% [67]. Blimp1-positive cells considered the progenitors of PGCs in both sexes, were expressed persistently from E6.25 to E13.5 [68,69]. Blimp1-Cre was found to be activated at E7.8 in PGCs, with a positive ratio of 55−78% [68]. However, as Blimp1 is also expressed in the retina, limbs, heart, pharynx, and B and T lymphocytes [70], Blimp1-Cre may not be suitable for all functional experiments. Tamoxifen-inducible ubiquitin C (UBC)-CreER^T2 [71,72] and ROSA26-CreER^T2 [73] were utilized for special target ablation in adult spermatogonia post-tamoxifen administration, showing high efficiency without detectable Cre recombination activity in somatic cells of the testis and other germ cells. Nonetheless, UBC-CreER^T2 activity was found in the somatic cells of 4-week mice testes, indicating an age-dependent Cre recombinase property [74,75]. Considering the broad expression of UBC and ROSA26, the predicted SSC phenotypes may be affected by functional defects in other tissues. Cre fused with the mouse mutant ER ligand-binding region is known as MerCreMer [76]. Oct-4 in PGCs starts at E7.5 in developmental embryos [77]. The Oct4-MerCreMer mouse line was used to perform the genetic analyses of undifferentiated spermatogonia, including As, Apr, and Aal types [78,79]. However, because Oct4 is expressed in various tissues during postnatal development, such as the skin, liver, and pancreas [80], the cKO phenotype should be evaluated to exclude other disturbances.

3. Cre Recombination Lines for Spermatocytes

3.1. Sycp1-Cre

Synaptonemal complex protein 1(SYCP1), primarily constituting the synaptonemal complex in meiosis, mainly functioned in recombination and XY body formation during leptotene to early pachytene stages [81]. Vidal et al. generated Sycp1-Cre mouse lines (Strain #:003466) and verified the recombination efficiency in mice harboring Sycp1-Cre and another transgene flanked by LoxP. They found that descendants of male double-transgenic mice mating with normal females were widely excised from the LoxP-flanked sequence. Even males were hemizygous for Sycp1-Cre, with the targets being removed in offspring without the Cre gene, indicating that recombination occurred during paternal spermatogenesis [16]. Additionally, the Sycp1-Cre driving target KO was restricted in the testis exclusively, and the flox-flanked segments were never altered in female progeny [16], likely due to the heterotopic transgenic expression initiated by partial promoters of Sycp1. Sanny et al. also confirmed that the specificity of Sycp1-Cre was restricted in the testes by crossing with R26R reporter transgenic mice, with recombination occurring in zygotene spermatocytes. Higher KO efficiency was observed in the entire meiosis stage, indicating that the Sycp1-Cre line is suitable for gene analysis during the germinal differentiation process [82].

Sycp1-Cre recombination activity decreased in the second generation during meiosis. This defect was associated with cytosine methylation, occurring in LoxP and transgenic sequences and extending to longer sequences in chromosomes. The allelic locus was also affected by a structure similar to the transvection defined in Drosophila [22]. In addition, Reza et al. created germ cell lines that were conditional knocked-out Ikkβ through mating with Sycp1-Cre mice; however, approximately 43% of the LoxP-flanked sequence was recombinant in offspring due to the epigenetic modification of LoxP in fourth- and fifth-generation (F4 and F5) mice [83]. The sodium bisulfite treatment sequencing revealed that methylated LoxP site cytosines were only found in F4 or F5 mice rather than in re-derived F1 Ikkβ^fl/fl mice unmated with Sycp1-Cre. The methylated LoxP sites were derived from Sycp1-Cre-sired IKKβ-LoxP alleles in paternal lines, while maternal LoxP was unmethylated [83]. Despite its suitability for gene analysis during germinal differentiation, Sycp1 presents epigenetic modifications and low recombination efficiency that cannot be neglected.

3.2. Prl3b1-Cre

Prolactin family 3, subfamily B, member 1 (Prl3b1) is localized in mouse chromosome 13, and its expression has been detected in the nervous system, eye, and epithelium of the digestive tract in mid-to-late developing embryos [84]. Prl3b1 is found in testicular germline cells, particularly in spermatocytes and haploid spermatids [85]. The Cre recombinase mouse line under the control of a 2.5 kb Prl3b1 promoter (Prl3b1-Cre) was generated, and efficiency and specificity analyses were conducted by mating with R26GRR mice expressing the fluorescent protein, tDsRed, after Cre activation [85]. Cre recombination activity was determined to be limited to the testis, epididymis, and seminiferous ducts. A strong fluorescence signal was observed from elongated spermatids and spermatozoa in seminiferous tubules, with 74% recombination efficiency detected in germ cells after in vitro fertilization [85]. In summary, the Prl3b1-Cre strain offers a powerful tool for investigating the genes involved in the spermatogenic process, owing to its robust specificity in germ cells, which is superior to that of the cKit-Cre, Pgk2-Cre, Hspa2-Cre, and Syn-Cre mouse strains reviewed in detail by Lee Smith [19] (Table 1).

3.3. Wisp3-Cre

Wisp3, activated by the Wnt-1 signaling pathway, is involved in multiple cellular physiological functions and carcinogenesis, including colon and breast cancers. Its mutation is related to progressive pseudorheumatoid arthritis, which is a type of autosomal recessive hereditary disease [86]. However, the mutation or overexpression of Wisp3 in mice results in no visible phenotypes compared to wild-type animals [87]. To further investigate the physiological actions and spatial expression of Wisp3 in mice, the Wisp3-GFPCre KI mouse line was established, with GFPCre inserted into the first exon of Wisp3, replacing Wisp3 expression with GFP and Cre. Cre recombination activity has been reported by mating with females carrying the ROSA26^mTmG allele. Higher levels of Cre-mediated recombination were observed only in the testes, especially during the prophase of meiosis I in spermatocytes. The recombinant offspring accounted for 7% of those born to females with two double heterozygotes with no recombination activity in the ovary [88]. In addition, William et al. concluded that Aurora A Kinase (AURKA) plays a role in spermatid physiology and mouse fecundity using the Aurka spermatocyte KO mouse strain, generated by crossing Aurka^fl/fl female mice with one floxed allele with Wisp3-Cre transgene male mice [89]. In conclusion, although Wisp3 is not essential for mouse fertility, the male mice carrying the Wisp3-Cre allele contribute to exploring the role of genes in the meiosis I stage of spermatogenesis.

3.4. Spo11-Cre

DNA double-strand breaks (DSBs) are crucial for initiating meiotic recombination and are initiated by SPO11. Spo11 mutation causes a synaptic defect in pachytene and leads to meiotic arrests in both males and females, resulting in the apoptosis of spermatocytes and oocytes [90]. Manuela et al. determined the physiological function of JAM-C in the germ cells of a cKO model in transgenic mice expressing IRES-Cre driven by Spo11 during early meiosis [91]. Lyndaker et al. also generated Spo11-IRES-Cre mouse tools (Strain #:032646) to investigate the role of HUS1 in DSB repair during meiotic prophase I (Figure 2) [92]. However, the transgenic Spo11 locus not only includes the entire Spo11 promoter sequence but also fuses with the IRES sequence upstream of the start site [91,92]. Further studies found that the expression of Spo11 in transgenic mice was disrupted by negative feedback, leading to a significant reduction in the Spo11 level and a direct and substantial impact on the meiotic process [93]. The Jordan group found that the main defect in spermatogenesis occurs in spermatocytes rather than in spermatids after Aurka mutation in Spo11-IRES-Cre cKO models [92,94]. They did not note that endogenic Spo11 function is disturbed by Cre recombination, which differs from the results obtained by William et al., who concluded that Aurka is required not only for spermatocyte maintenance but also for spermatid morphogenesis using Aurka spermatocyte KO mice excised by Wisp3-Cre [89]. In summary, the use of Spo11-Cre in the study of gene functions in spermatocytes is limited and has rarely been reported.

4. Cre Recombination Strains for Spermatids

4.1. Tspy-Cre

Human Tspy, localized on the Y chromosome, encodes proteins expressed in the testis and is conserved in placental mammals, including artiodactyl [95], rodents [96], and perissodactyl [97]. Tspy functions in the cell cycle with differentiation, indicating its role in spermatogonial development [98]. As Tspy was silenced naturally in experimental mice, human Tspy, including a 2.8 kb coding region with a 2.95 kb promoter region, was used to establish a transgenic mouse line to analyze Tspy physiological characteristics in the testis [99]. Mice harboring human Tspy (hTspy) presented normal phenotypes, and the counts of pachytene spermatocytes and spermatids did not change sharply compared to wild-type controls [99]. Furthermore, the hTspy-Cre recombinant mouse was constructed in the control of a 2.4 kb hTspy promoter, and it was found that the hTspy recombination activity was mainly in round and elongated spermatids, as shown by EGFP immunostaining in the progenies born from double hTspy-Cre/Z/EG mice expressing EGFP, especially in the testis [100]. Additionally, EGFP is expressed in the ovary and in the central and peripheral nervous systems as early as E12.5. Thus, the hTspy-Cre transgenic mouse line can be used as a model for exploring gametogenesis. However, Cre activation in multiple tissues may result in an inaccurate determination.

4.2. Prm1-Cre

Endogenous protamine is expressed in the haploid stages of spermatogenesis [101], and exogenous genes fused with the Prm1 proximal promoter are primarily limited to haploid spermatids [102,103] despite lower ectopic expression in the heart and temporal bone [104]. One type of Prm1-Cre mouse line (Strain #:003328) comprises a fusion of Cre with the 652 bp fragment of the mouse Prm1 promoter. Recombination efficiency was primarily observed in male germ cells with non-significant functionality both in embryonic stem cell lines and somatic tissues from embryos or adult mice [17]. However, the recombination efficiency of Prm1-Cre is doubtful, with only about 50% reported when flox-flanked Pofut1 and Mgat1 were silenced, as noted by Frank et al. [23]. Another Prm1-Cre mouse line developed by Schmidt et al. demonstrated the catalytic activity of Prm1-Cre recombinase in post-meiotic spermatids. However, all paternal Cre-bearing mice and Cre-modified male offspring modified by Cre from female founders were infertile. Further analysis revealed that 100% of abortive pregnancies were caused by spermatid chromosome rearrangements catalyzed by Cre recombinase in embryos acquired from wild-type females crossed with males carrying the Cre gene [105].

4.3. Acrv1-iCre

Cre recombinase specific to spermatids was established to be limited to haploid cells after meiotic division. Offsprings from mating floxed transgenic and heterozygote Cre recombination strains are the result of an inevitable part of haploid sperm being recombination-deficient without exhibiting presumptive phenotypes. Theoretically, approximately half of non-recombining sperm hold some promise for normal reproduction. Thus, increasing recombination efficiency is essential for targeting genes of interest in spermatids. Recently, an Acrv1-iCre mouse line was genetically engineered using CRISPR/Cas9 to selectively excise genetic segments in spermatids [106]. The expression of iCre, initiated by the promoters of Acrv1, which is specifically localized in stage 5−8 spermatids, efficiently deleted the floxed allele by more than 97%, as quantified by assessing the percentage of progeny with intact floxed alleles or deletion upon the mating type of Rosa26^Acrv1-iCre/+ with Dot1f^fl/fl [106]. The generation and characterization of the Acrv1-iCre recombination line by Julie et al. [106] contradicted the hypothesis that a small number of alleles in haploid spermatids would not be knocked out because of the common crossing approach. We assumed that Cre transcripts already functioned either prior to or during the second meiotic division process. Cre-negative spermatids can activate recombination activity through intercellular communication, such as exocytosis [107], which coincides with the concept that Cre can pass between haploid spermatozoa via cytoplasmic bridges [19]. Moreover, Cre transgenic mouse lines offer an advantageous technique for establishing global KO models to investigate large quantities of gene sequences in somatic tissues if normal reproductive behavior is observed after the genes in either early germ cells or haploid spermatozoa are excised.

4.4. Elf5-Cre

ELF5, an ETS transcription factor, is localized in the trophoblast lineages of the embryo as well as in the prostate, kidney, lungs, testes, and mammary gland during postnatal development [108,109,110]. Shuangbo et al. established an Elf5-Cre transgenic mouse line by co-injecting the constructed 2A-Cre coding sequence with Cas9/sgRNA into the pronuclei, and the Cre sequence was integrated into the exon of Elf5 close to the stop codon to investigate the contribution of genes in placental development (Figure 2) [108]. Typically, Cre is activated in all trophoblast-derived lineages. Furthermore, a robust signal was detected in spermatids and sperm, suggesting a novel transgenic mouse strain for functional gene studies confined to the late stage of spermatogenesis [108]. However, due to the catalytic activity of Elf5-Cre, attention should be paid to its specificity and efficiency when studying genes of interest in haploid sperm.

Table 1. Characteristics of Cre models generated for male germline research.

Marker Strains	Germline Specific	Expression Outside of the Reproductive System	Initial Expression Phase	Transgenic (Tg)/Knock-In (KI)
Oct4-MerCreMer [78,79] (Strain #016829)	PGCs and undifferentiated spermatogonia	Pancreas, skin, intestine, kidney, etc. [80]	E7.5–8 [77]	KI
Tnap-Cre [66]	PGCs (around 50%)	Placenta, intestine and neural tube, labyrinthine region	E9.5–10.5	KI
Nanos3-Cre [67]	PGCs (11–25%)	NR	E7.75	KI
Nanos2-MerCreMer [111,112]	undifferentiated spermatogonia	NR	E13.5 [113]	Tg
Blimp1-Cre [68,114] Strain #008827	PGCs (55–78%)	B and T lymphocytes, retina, limbs, pharynx, and heart [70]	E6.25	Tg
Tex101-iCre [115,116] Strain #019893	Pro-spermatogonia and subsequent germ cells	NR	1 DPP	Tg
Gfra1-CreERT2 [117]	Undifferentiated spermatogonia	Kidney [118]	E9.5	KI
UBC-CreERT2 [71,72,74,75,119] Strain #:007001	Spermatogonia, testis, and somatic cells	Thymus, spleen, heart, muscle, brain, kidney, bone marrow [119]	NR	Tg
Rosa26-CreERT2 [73]	Spermatogonia	Other tissues in embryo and adult [120]	NR	KI
Aqp2-Cre [121] Strain #:006881	Spermatids	Kidney	NR	Tg
Hspa2-Cre [122,123] Strain #:008870	Spermatocyte and spermatids	Brain and embryo	Leptotene	Tg
Pgk2-Cre [124,125]	Spermatocyte and spermatids	Tissues in embryo [125]	NR	Tg
Wnt7a-Cre [126,127] Strain #036637-JAX	Spermatocyte	Uterine epithelium [127]	Mid-pachynema (12 DPP)	Tg
cKit-Cre [128]	Spermatocytes and spermatids	Mosaicism (20–100%)	NR	Tg
CaMKIIα-Cre [129,130] Strain #:005359	Testis germ cells	Brain	NR	Tg
Syn1-Cre [131,132] Strain #:003966	Spermatocytes	Neurons [132]	E12.5	Tg

NR: not reported.

Table 1. Characteristics of Cre models generated for male germline research.

Marker Strains	Germline Specific	Expression Outside of the Reproductive System	Initial Expression Phase	Transgenic (Tg)/Knock-In (KI)
Oct4-MerCreMer [78,79] (Strain #016829)	PGCs and undifferentiated spermatogonia	Pancreas, skin, intestine, kidney, etc. [80]	E7.5–8 [77]	KI
Tnap-Cre [66]	PGCs (around 50%)	Placenta, intestine and neural tube, labyrinthine region	E9.5–10.5	KI
Nanos3-Cre [67]	PGCs (11–25%)	NR	E7.75	KI
Nanos2-MerCreMer [111,112]	undifferentiated spermatogonia	NR	E13.5 [113]	Tg
Blimp1-Cre [68,114] Strain #008827	PGCs (55–78%)	B and T lymphocytes, retina, limbs, pharynx, and heart [70]	E6.25	Tg
Tex101-iCre [115,116] Strain #019893	Pro-spermatogonia and subsequent germ cells	NR	1 DPP	Tg
Gfra1-CreERT2 [117]	Undifferentiated spermatogonia	Kidney [118]	E9.5	KI
UBC-CreERT2 [71,72,74,75,119] Strain #:007001	Spermatogonia, testis, and somatic cells	Thymus, spleen, heart, muscle, brain, kidney, bone marrow [119]	NR	Tg
Rosa26-CreERT2 [73]	Spermatogonia	Other tissues in embryo and adult [120]	NR	KI
Aqp2-Cre [121] Strain #:006881	Spermatids	Kidney	NR	Tg
Hspa2-Cre [122,123] Strain #:008870	Spermatocyte and spermatids	Brain and embryo	Leptotene	Tg
Pgk2-Cre [124,125]	Spermatocyte and spermatids	Tissues in embryo [125]	NR	Tg
Wnt7a-Cre [126,127] Strain #036637-JAX	Spermatocyte	Uterine epithelium [127]	Mid-pachynema (12 DPP)	Tg
cKit-Cre [128]	Spermatocytes and spermatids	Mosaicism (20–100%)	NR	Tg
CaMKIIα-Cre [129,130] Strain #:005359	Testis germ cells	Brain	NR	Tg
Syn1-Cre [131,132] Strain #:003966	Spermatocytes	Neurons [132]	E12.5	Tg

NR: not reported.

5. Cre Transgenic Mice for Sertoli and Leydig Cells

Cre lines specific for Sertoli cells, including Abp-Cre, Amh-Cre (Strain #:007915), Dhh-Cre (Strain #:012929), and Dmrt1-Cre, have been used extensively and were described by Smith [19]. Here, novel Cre lines specific to Sertoli cells and their practical applications are described. Sry was exclusively detected in the supporting cells of genital ridges during E10.5–E12.5 and mediated the fate of supporting cells towards Sertoli cells [133]. Sry-Cre compromising the 9.9 kb Sry sequence under the controls of 5′ and 3′, untranslated regions of endogenous Sry, was constructed to explore the fate of Sry-positive cells [134] as well as the functional region of sex determination-related genes [135,136]. Sox9 is expressed in multiple tissues and cells, including the CNS, intestine, and Sertoli cells, and regulates cell growth and differentiation during mouse embryogenesis. The Sox9-Cre transgenic line was generated by fusing Cre recombinase with an internal ribosome entry sequence and knocking it into the Sox9 locus at the 3′ untranslated region [137]. The systematic analysis of Sox9-Cre/R26R mice revealed that Sox9-positive cells, as progenitors, were conducive to a variety of cell types, including chondrocytes, Leydig cells in the testis, intestinal epithelial cells, and all cells in the pancreas and spinal cord of the mouse embryo from E8 to E17 [137]. Therefore, the Sox9-Cre mouse line can be used for sex differentiation investigations because of its catalytic activity in early embryos. Yayoi et al. established a Sox9-Cre/Nr5a1^fl/fl mouse strain and concluded that Nr5a1 plays a crucial role in mouse gonadal sex determination [138]. Sox9-CreER mice (Strain #:035092) were generated after IRES-CreER^T2-SV40pA cassettes integrating into endogenous Sox9 at 3′UTR by the Cas9/RNA targeting method and have been applied in embryonic lineage tracing experiments after mating with Rosa-stop-mTmG mice [139]. However, Sox9-Cre was restricted in its application to males due to its recombinant activity in a broad range of tissues.

Cyp17-iCre (Strain #:028547) and Cyp11a1-iCre in Leydig cells were reviewed in 2011 with ectopic expression in the brain and adrenal glands [19]. Additionally, a second Cyp11a1-iCre transgenic line was generated by BAC construction comprising a 2.8 kb Cyp11a1 and iCre sequence, and the iCre catalytic activity was controlled by the mouse promoter [140]. Laura et al. established another novel mouse line, Cyp11a1-GC, with dual characteristics: it not only silenced the endogenous Cyp11a1 function but also simultaneously knocked-in Cre recombinase, which excises the genes of interest in steroidogenic cells without changes in the circulating testosterone concentration [141]. Annalucia et al. characterized the ability of viral vectors, including adenovirus, lentivirus, and adeno-associated virus (AAV), to deliver exogenous genes targeting Leydig cells in adult mouse testes and determined that AAV serotype 9 (AAV9) + neuraminidase transported the transgenes efficiently [142]. Moreover, AAV9-driven Cre was generated by the Diane group to delete endogenous glucocorticoid receptors in adult Leydig cells; the silencing efficiency of AAV9-Cre to AR was 48% 7 days after injection, which was higher than that of the Cyp17a1-iCre transgenic approach (28%) [143]. The AAV9 virus was found to infect germ cells of the testes from 3-week mice [144], possibly because these testes are not fully developed in immature mice, and the blood–testis barrier is not fully established. Thus, the AAV9-Cre silencing method is more suitable for exploring the genetics underlying the functions of adult Leydig cells, whereas the Cyp17a1-iCre model is more suitable for functional studies of developing Leydig cells.

The type II Amh receptor, Amhr2, is localized in the mesenchyme of the Müllerian duct, Leydig cells, Sertoli cells, and granulosa cells and initiates the degeneration of Müllerian ducts after binding with the anti-Müllerian hormone [145]. The Amhr2-Cre line (B6;129S7-Amhr2^tm3(cre)Bhr/Mmnc from the Mutant Mouse Regional Resource Centers) was generated by knocking in the targeting vector to the endogenous Amhr2 loci (Figure 2) [146]. The catalytic activity of Amhr2-Cre was observed in both the Leydig and Sertoli cells of the testis, theca cells, and granulosa cells in the ovary [147,148]. The Amhr2-Cre mouse model has also been used as a genic toolkit for the study of testicular granulosa cell tumors [149]. Notably, Amhr2-Cre mice mating with (translocator protein) Tspo-floxed mice generated global Tspo KO mice instead of the Tspo cKO line [24], probably because of the genetic linkage of Amhr2 with Tspo, which are expressed together during the early embryo stage. Thus, emphasis should be placed on whether global KO occurs when using the Amhr2-Cre mutation approach. Furthermore, the Chauvin group, collaborating with the Jackson Laboratory, developed Amhr2-CreER^T2 mice (Strain #:037056) by the CRISPR/Cas9-mediated approach, which has been explored in determining the fate of cancer-associated mesothelial cells in ovarian cancer upon Amhr2 induction by crossing with the ROSA26^mTmG mouse line [150]. The recombination efficiency of this novel mouse line requires further evaluation.

6. Cre Transgenic Models for Other Cells in the Testes

In addition to spermatogenic, Leydig, and Sertoli cells, various other cell types have been well established in the testes, including T cells, endothelial cells, peritubular myoid cells, mesenchymal (stromal) cells, macrophages, tenocytes, two pericyte subpopulations (with either smooth muscle or ECM-secreting properties), and a Leydig cell precursor population [151,152,153]. However, the functional characteristics of these cells contributing to spermatogenesis are yet to be elucidated, and their protein expression patterns have not been identified. Several generalized Cre lines were used to investigate the gene function in these cells. smMHC-Cre (also called Myh11-Cre) (Strain #007742) for vascular smooth muscle cells and peritubular myoid cells (PTM) and SM22-Cre lines (Strain #:004746) for PTM were described by Smith [19]. Myh11-Cre was used to uncover the multiple functions of genes in testicular PTM. The synthesis of the testosterone-dependent glial cell line-derived neurotrophic factor (GDNF) in PTM cells was found to maintain the microenvironment of the spermatogonial stem cell (SSC) niche, together with Sertoli cells [154]. The initial recombination activity of the Myh11-Cre mouse line was detected at E12.5, and Cre efficiency was observed in all smooth muscles of adult mice, including the heart, bladder, lung, and testes [155]. The essential roles of GDNF [156] and Gops5 [157] in PTM cells on mice reproductive function and postnatal development in PTM cells have been demonstrated in Myh11-Cre cKO models. Furthermore, tamoxifen-inducible Myh11-CreER^T2 or Myh11-DreER^T2 mouse strains virtually eliminated specific targets in PTM cells [158]. However, Myh11-Cre expression in the testes was not restricted to PTM cells and was also found in blood vessels; thus, interference may occur in the prospective phenotype in cKO mice [159]. Moreover, Cxc3 chemokine receptor 1 (Cx3cr1) was found in monocytes and resident macrophages in all mouse tissues [160]. Cx3cr1-Cre mice were generated and used for the conditional expression of the diphtheria toxin receptor in testis macrophages for further excision, despite no direct evidence of a cKO event, with an efficiency of approximately 95%, indicating the effectiveness of Cx3cr1-Cre in the macrophage population [160,161]. However, Cx3cr1 is extensively expressed in macrophages and monocytes; therefore, potential disturbances cannot be ignored.

Evidence suggests that a Cre line is generated, particularly targeting endothelial cells of the testis. Tie2-Cre (Strain #:008863) is localized in endothelial cells in the testis [19,162]. O’Hara et al. performed further research by applying Tie2-Cre to inactivate androgen receptors in testicular vascular endothelial cells [163]. Additionally, Cre lines that have undergone careful validation in other tissues are expected to remove the targets of interest in endothelial cells of the testis, such as Cdh5-Cre [164] and Pdgfb-iCreER^T2 [165]. Concerns regarding Cre-engineered mouse strains, especially macrophages, have been widely reported, with LysM-Cre and hCD68-CreER^T2 being the common methodologies [166]. LysM-Cre is prominently expressed in major myeloid lineage cell types, such as monocytes and neutrophils (Figure 2), whereas hCD68-CreER^T2 primarily targets tissue-resident macrophages and is inducible by tamoxifen [167]. LysM-Cre was utilized to generate myeloid-specific ubiquitin-specific protease 2 (USP2) cKO mice, revealing the necessity of macrophage USP2 for sperm physiological functions, including motility, capacitation, and hyperactivation [168]. Despite reports of the recombination efficiency of Cre, evidence supporting its ability to eliminate specific genes in testicular macrophages remains lacking. Other cell types identified through single-cell sequencing in the testes, such as T cells, mesenchymal (stromal) cells, and tenocytes [151,152,153], have been poorly studied in terms of their regulation of germ cell development, and a detailed description of the cKO strategy is not provided.

7. Cre Models for Epididymis

Sperm exhibit progressive motility and fertilization properties during transit in the epididymis, a highly convoluted duct comprising four main anatomical parts as follows: the initial segment (IS), caput, corpus, and cauda, each with distinct regional functions and characteristics [169]. The microenvironment of the epididymal lumen undergoes various changes during sperm transportation, with a large number of ions, proteins, and miRNAs absorbed or released into the lumen fluid [169]. Approximately 40% of male idiopathic infertility cases are associated with defects in sperm maturation, underscoring the importance of the epididymal function in sperm maturation [170]. Although assisted reproductive techniques (ART) contribute to improving sperm maturation status, reproductive risks related to artificial interventions are increasingly emphasized [170]. It is necessary to elucidate the mechanism of sperm maturation in the epididymis and ameliorate male reproductive performance with minimal external interventions. To date, multiple Cre- engineered mouse models have been employed in many investigations of targets in the epididymal segments.

7.1. Defb41-iCre

DEFB41, containing 62 amino acids, is a specific beta-defensin, primarily localized in the epithelial cells of the initial segment and caput, with weak signals in the prostate, corpus, and pancreas [171]. The initial expression was detected at 7−14 DPP, peaked at 25 DPP, and remained stable after 40 DPP [172]. The Defb41-iCre KI mouse line was generated using the red/ET recombination approach, and the first exon of Defb41 was inserted with Cre recombinase constructed in a BAC [172]. Björkgren et al. showed that Defb41 ablation in the epididymis altered sperm progressive motility and the ability to bind to the oocyte, whereas sperm morphology and count were unaffected in the homozygous Defb41^iCre/iCre mouse strain [172]. Consequently, heterozygous Defb41^iCre/+ mice were used as a cKO tool line to detect Dicer1 physiological properties in the initial segment and caput by the progeny from Dicer1^fl/fl; Defb41^iCre/+ mice [173,174].

7.2. Rnase10-Cre

The initial expression of Rnase10, coinciding with IS differentiation, was detected at approximately 17 DPP [175]. The disruption of proximal proteins encoded by Rnase10 in the mouse epididymis is associated with a penetrating defect in the zona pellucida, rendering sperm unable to pass through the female uterotubal junction district [176]. The Rnase10-iCre line was established by introducing the iCre-NeoR cassette into the Rnase10 translation initiation locus in the first eight nucleotides of exon 2 [177]. The androgen receptor (AR) was inactivated by the Rnase10-iCre mice mating with AR^fl/fl homozygous females, validating the critical role of AR in the function and development of IS [177].

7.3. Crisp4-Cre

CRISP4 is a cysteine-rich secretory protein (CRISP) that is highly expressed in murine principal cells in the epididymal epithelium, with its transcripts most abundantly present in the caput and corpus and few signals in the thymus and spleen [178,179]. CRISP4 has also been detected in epididymosomes and seminal plasma [180] and is strongly associated with sperm maturation during the epididymal transport process [181]. CRISP4 inactivation causes the failure of protein tyrosine phosphorylation and the acrosome reaction induced by progesterone in the capacitation process [182] and is incapable of fertilization with zona pellucida-intact eggs, probably due to the calcium channel TRPM8 defect and failure to regulate the acrosome reaction [181]. Crisp4-iCre KI mice were generated by inserting the iCre-neomycin phosphotransferase cassette into the third Crisp4 exon locus before the initiation codon, resulting in a transcription frameshift. The homozygous iCre-recombinase line acts as a Crisp4-deficient model, and the heterozygous mouse model can be used to excise targets of interest in the epididymis [180]. Crisp4-iCre is expressed in the epididymis at day 20 of postnatal development, and its level increases with age [180]. The specificity of the Crisp4-iCre recombination event performed by Crisp4^+/−/Z/RED⁺ and Crisp4^+/−/Runx1^fl/+ transgenic mice was expressed in the whole epididymis tissue and in proximal epididymis without signals in other tissues detected, respectively [180], indicating that Crisp4-iCre is capable of ablating targets in vivo.

7.4. Lipocalin-Cre

mE-RABP (Lcn5), mEP17 (Lcn8), and lipocalin 9 (Lcn9), defined in the lipocalin family by phylogenetic analysis, are murine secretory proteins localized in the epididymis [183]. Lcn5 is synthesized in principal cells residing in the middle/distal caput regions and is initially secreted from 30 DPP, with a gradual increase to 60 DPP [184]. Lcn8 and Lcn9 are positioned in the IS with similar expression patterns and are expressed at 21 DPP during postnatal development, depending on testicular factor regulation [183,185]. Spermatogenesis and fertility were normal conditions after Lcn8 or Lcn9 inactivation in the epididymis, while Lcn8 ablation caused an increased teratospermia rate, sperm motility, and acrosome reaction frequency deficiency, indicating its indispensable role in sperm maturation [186]. Lipocalin family genes are highly conserved and show homology among subtypes, with the probability of functional overlap in a physiological manner. The transgenic lines in Lcn5, Lcn6, Lcn8, and Lcn10 were knocked out simultaneously, or Lcn5, Lcn6, Lcn8, Lcn10, and Lcn9, when silenced synchronously, showed subfertility and infertility in most cases [187].

The Lcn5-Cre transgenic mice were established by Xie et al. using the Cre/LoxP system, where Cre activity was regulated by the Lcn5 promoter (1.8 kb). Initially, Cre catalytic activity was observed at 30 DPP, showing high specificity in middle or distal caput principal cells when crossed with the reporter strain mT/mG or with the Aip1^fl/+ mouse line. The recombination efficiency was statistically significant at 28.9% [188]. A tamoxifen-sensitive Lcn5-CreER^T2 transgenic line was generated by the same group. Cre activity was also highly restricted in the caput epididymis by tamoxifen induction in a time- and dose-dependent manner, providing an approach for the further analysis of the spatiotemporal functions of target genes in the caput epididymis [189]. Lcn8-Cre or Lcn9-Cre mice were generated by the insertion of the NLS-Cre-polyA or 2A-NLS-Cre cassette into the Lcn8 or Lcn9 promoters, respectively, using CRISPR/Cas9 technology; Cre expression led to the loss of Lcn8 and Lcn9. Lcn8-Cre and Lcn9-Cre activity is specially confined to the principal cells of the IS, without reproductive disorders, in adult male mice [190,191]. Novel Lcn8-Cre and Lcn9-Cre models can be used to conduct functional gene studies that are specific to IS segments (Figure 2). However, transgenic mouse models with Cre bioactivity in the epididymal cauda are still lacking; thus, generating more recombinant mouse lines, particularly those with silencing targets in the epididymis, is necessary.

8. The Cre Models Generated for the Prostate, Seminal Vesicle, and Seminiferous Duct

The prostate is an adnexal gland of the male genitourinary system, primarily comprising the stroma and epithelium. Prostatic fluid is secreted by the prostate epithelial compartment and occupies approximately 1/5 to 1/3 of the volume of ejaculated semen. It contributes to sperm motility, the clotting cycle, and semen liquefaction [192]. Cre expression under the regulation of the promoter derived from the prostate-specific probasin (ARR2PB) of rats and ARR2PB-Cre (Strain #:026662) catalytic activity was detected predominantly in all lobes of the mouse prostate. The highest signals were observed in the lateral lobes driving androgen-dependent transcription events, while Cre activity was least visible in the anterior and dorsal lobes. The further functional characterization of ARR2PB-Cre was carried out by selectively removing RXRa from the mouse prostate and avoiding the embryonic lethality caused by RXRa global KO [193]. In addition, Jin et al. generated an ARR2PBi-Cre transgenic mouse line (Strain #023325), showing uniform expression across all lobes of the prostate as well as in the ductus deferens and seminal vesicles. This model exhibited higher efficiency in interfering with gene activity in target tissues [194]. ARR2PBi-Cre mice are a valuable tool for genetic-based investigations of the prostate, seminal vesicles, and ductus deferens (Figure 2).

9. Conclusions and Perspective

An orchestrated series of events, mainly involving PGC development, SSC maintenance, spermatogenesis, sperm maturation, and ejaculation, ensures male fertility under normal conditions depending on certain genetic expressions during different physiological phases. Conventional KOs often lead to embryonic or perinatal lethality, particularly in homozygotes, and may result in unpredictable impacts on other organs with abnormal physiology. The increasing use of Cre recombination lines highlights the validity of the Cre/LoxP system as a valid molecular genetics approach for reproductive research. In this study, we summarized the Cre mouse lines used to investigate gene behavior during various processes related to germ cell biology and sperm physiological function. We also addressed existing issues such as unstable efficiency, global KO, the ectopic expression of Cre activity, and strong maternal effects. The unpredictable expression pattern of Cre in the testes may be attributed to chromatin compaction and rearrangement during meiosis, as well as the randomness of insertion sites and cassette defects in traditional gene recombination methods. As early as 2013, the CRISPR/Cas9 system was used to mutate the mouse genome, resulting in a genetically disrupted mouse line that was achieved faster and at lower costs [13]. CRISPR/Cas9 can act on multiple loci simultaneously and contribute to investigating the combined effects of genes on reproduction or establish connections among targeted genes in the fertility process [195,196]. Moreover, random insertions and uncontrolled gene copies caused by transgenic steps in conventional recombination are avoided in the CRISPR/Cas9 system [197]. In this review, we summarized several gene-disrupted mouse lines edited by the CRISPR/Cas9 approach, including Ddx4^iCre [15], Ddx4^{em1(CreERT2)Utr} [20], Stra8^P2A-Cre [43], Acrv1-iCre [106], Lcn8-Cre and Lcn9-Cre [190,191], which showed higher specificity, efficiency, and the lower occurrence of global KO events. Other recommendations are as follows: 1. Combining multiple inducible Cre lines can help inactivate target genes localized in distinct regions of the epididymis, as some genes are distributed throughout the entire epididymis without regional differences, and a clear phenotype may not be visible under gene disruption in a single segment. 2. The strict control of Cre-LoxP can be strengthened by combining it with the Dre-Rox system, making lineage tracing more precise and minimizing the non-determinate effects of Cre catalytic activity on certain cells [198]. Thus, a more credible and accurate in vivo study of the cell lineage from the male reproductive system is warranted by this novel technology, as it greatly reduces the nonspecific recombination of the conventional Cre-LoxP system. 3. There is a demand to generate new homozygous Cre lines for gene recombination in spermatids with higher efficiency and stable outcomes, and it is worth considering the expansion of Cre models for the corpus and cauda epididymis.

Overall, as a large number of genes play a vital role during the male reproductive process, more mouse tool models can be established and be made available for detailed descriptions of genes to reveal the male reproductive mystery at the genetic level.