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Retinoic Acid and Germ Cell Development in the Ovary and Testis
卵巢和睾丸中的视黄酸和生殖细胞发育

by 1,*,
作者: 1,*
2,†,
1,* 2,†
2,†,
2,† 2,†
2,3,4,* and
2,† 2,3,4,*
2,5,6,*
2,3,4,* 2,5,6,*
1
Immunology Frontier Research Center, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita 565-0871, Japan
大阪大学微生物病研究所免疫学前沿研究中心,日本吹田县山道县 3-1 565-0871
2
Whitehead Institute, Cambridge, MA 02142, USA
怀特黑德研究所, 剑桥, 马萨诸塞州 02142, 美国
3
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
麻省理工学院生物系, 剑桥, 马萨诸塞州 02139, 美国
4
Howard Hughes Medical Institute, Whitehead Institute, Cambridge, MA 02142, USA
霍华德休斯医学研究所,怀特黑德研究所,剑桥,马萨诸塞州 02142,美国
5
Reproductive Biology Group, Division of Developmental Biology, Department of Biology, Faculty of Science, Utrecht University, 3584 CH Utrecht, The Netherlands
乌得勒支大学理学院生物系发育生物学部生殖生物学组,3584 CH Utrecht, The Netherlands
6
Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands
阿姆斯特丹大学学术医学中心生殖医学中心,1105 AZ 阿姆斯特丹,荷兰
*
Authors to whom correspondence should be addressed.
应向其发送信件的作者。
These authors contributed equally to this work.
这些作者对这项工作做出了同样的贡献。
Biomolecules 2019, 9(12), 775; https://doi.org/10.3390/biom9120775
生物分子 2019, 9(12), 775;https://doi.org/10.3390/biom9120775
Submission received: 31 October 2019 / Revised: 22 November 2019 / Accepted: 23 November 2019 / Published: 24 November 2019
收到意见书:2019 年 10 月 31 日 / 修订日期:2019 年 11 月 22 日 / 接受日期:2019 年 11 月 23 日 / 出版日期:2019 年 11 月 24 日
(This article belongs to the Special Issue Retinoids in Embryonic Development)
(本文属于胚胎发育中的类维生素 A 专刊)

Abstract 抽象

Retinoic acid (RA), a derivative of vitamin A, is critical for the production of oocytes and sperm in mammals. These gametes derive from primordial germ cells, which colonize the nascent gonad, and later undertake sexual differentiation to produce oocytes or sperm. During fetal development, germ cells in the ovary initiate meiosis in response to RA, whereas those in the testis do not yet initiate meiosis, as they are insulated from RA, and undergo cell cycle arrest. After birth, male germ cells resume proliferation and undergo a transition to spermatogonia, which are destined to develop into haploid spermatozoa via spermatogenesis. Recent findings indicate that RA levels change periodically in adult testes to direct not only meiotic initiation, but also other key developmental transitions to ensure that spermatogenesis is precisely organized for the prodigious output of sperm. This review focuses on how female and male germ cells develop in the ovary and testis, respectively, and the role of RA in this process.
视黄酸 (RA) 是维生素 A 的衍生物,对哺乳动物卵母细胞和精子的产生至关重要。这些配子来源于原始生殖细胞,这些细胞定植于新生的性腺,然后进行性分化以产生卵母细胞或精子。在胎儿发育过程中,卵巢中的生殖细胞响应 RA 启动减数分裂,而睾丸中的生殖细胞尚未启动减数分裂,因为它们与 RA 绝缘,并经历细胞周期停滞。出生后,雄性生殖细胞恢复增殖并过渡到精原细胞,精原细胞注定会通过精子发生发育成单倍体精子。最近的研究结果表明,成人睾丸的 RA 水平会周期性地变化,不仅指导减数分裂的开始,还指导其他关键的发育转变,以确保精子发生得到精确组织,以实现精子的惊人输出。本文重点介绍女性和男性生殖细胞在卵巢和睾丸中的发育方式,以及 RA 在此过程中的作用。
Keywords:
germ cells; retinoic acid; meiosis; ovary; spermatogenesis; testis
关键词:生殖细胞;视黄酸;减数分裂;卵巢;精子;在测试

1. Introduction 1. 引言

Mammalian oocytes and sperm derive from the same embryonic precursor cells, called primordial germ cells (PGCs). In development, PGCs migrate to the somatic gonad, where they undertake gametogenesis to ultimately produce oocytes or sperm, depending on whether they are in an ovary (female) or a testis (male). In mice, differences between the somatic cellular composition of ovaries and testes are microscopically evident by embryonic day (E) 12.5 [1,2,3]. Germ cells, however, remain morphologically indistinguishable between the sexes until E13.5 [4,5]. Subsequently, female germ cells enter meiotic prophase I and begin to differentiate as oocytes, whereas male germ cells remain mitotically active, and later arrest in the G0/G1 phase of the mitotic cell cycle [4,5].
哺乳动物卵母细胞和精子来源于相同的胚胎前体细胞,称为原始生殖细胞 (PGC)。在发育过程中,PGC 迁移到体细胞性腺,在那里它们进行配子发生,最终产生卵母细胞或精子,具体取决于它们是在卵巢(女性)还是睾丸(男性)中。在小鼠中,卵巢和睾丸的体细胞组成之间的差异在胚胎日(E)12.5的显微镜下很明显[1,2,3]。然而,生殖细胞在性别之间在形态上仍然无法区分,直到 E13.5 [4,5]。随后,雌性生殖细胞进入减数分裂前期 I 并开始分化为卵母细胞,而雄性生殖细胞保持有丝分裂活性,随后停滞在有丝分裂细胞周期的 G0/G1 期 [4,5]。
In the 1970s, Byskov and Saxen [6] suggested that a “meiosis inducing substance”, present in the embryonic ovary, is required for germ cells to initiate meiotic prophase. Recent studies find that retinoic acid (RA) is such a meiosis-inducing substance, produced by the somatic cells of the gonad and mesonephros [7,8,9]. RA is generated from dietary vitamin A (also known as retinol) by a series of oxidative reactions. Local levels of RA are regulated by retinaldehyde dehydrogenases, which catalyze the last step of RA synthesis, and by a cytochrome p450 enzyme (CYP26B1), which degrades RA [10,11] (reviewed in [12,13]). This metabolism of RA regulates whether female and male germ cells initiate meiosis in the fetal ovary, or the adult testis, respectively.
在 1970 年代,Byskov 和 Saxen [6] 提出,生殖细胞启动减数分裂前期需要存在于胚胎卵巢中的“减数分裂诱导物质”。最近的研究发现,视黄酸 (RA) 是一种诱导减数分裂的物质,由性腺和中肾的体细胞产生 [7,8,9]。RA 是由膳食维生素 A(也称为视黄醇)通过一系列氧化反应产生的。RA 的局部水平受视黄醛脱氢酶(催化 RA 合成的最后一步)和细胞色素 p450 酶 (CYP26B1) 的调节,后者可降解 RA [10,11](见 [12,13])。RA 的这种代谢调节女性和男性生殖细胞是分别在胎儿卵巢还是成人睾丸中启动减数分裂。
Male germ cells, which are arrested in G0/G1 phase of the cell cycle in the fetal testis, later resume proliferation and undergo a transition to spermatogonia after birth [14,15]. Spermatogonia, which include germline stem cells, undergo an elaborately organized process to give rise to specialized haploid gametes, called spermatozoa [16]. The complete process of germ cell development, from spermatogonia to spermatozoa, is called spermatogenesis. Within the testis, several developmental transitions of spermatogenesis, including spermatogonial differentiation and meiotic initiation, occur in close physical and temporal proximity. Over several decades, pharmacological and genetic studies have revealed that these key transitions are strictly regulated by RA [17,18,19]. In this review, we focus on how germ cell development is coordinated in the ovary and testis, and the instructive role of RA in this process.
雄性生殖细胞在胎儿睾丸细胞周期的G0/G1期被停滞,出生后恢复增殖并过渡到精原细胞[14,15]。精原细胞(包括种系干细胞)经历一个精心组织的过程,产生专门的单倍体配子,称为精子[16]。生殖细胞发育的完整过程,从精原细胞到精子,称为精子发生。在睾丸内,精子发生的几个发育转变,包括精原分化和减数分裂起始,在物理和时间上非常接近地发生。几十年来,药理学和遗传学研究表明,这些关键转变受到 RA 的严格控制 [17,18,19]。在这篇综述中,我们重点介绍生殖细胞发育如何在卵巢和睾丸中协调,以及 RA 在此过程中的指导作用。

2. Germ Cell Development in the Fetal Gonad
2. 胎儿性腺中的生殖细胞发育

Shortly after entry to the gonads, germ cells acquire the competence for meiotic initiation and sexual differentiation in the fetal gonad. Whether germ cells initiate meiosis or continue in a mitotic cell cycle is determined by their gonadal environment, rather than their sex chromosome constitution (reviewed in [20]). Germ cells in the fetal ovary are exposed to RA and initiate meiosis, whereas those in the fetal testis are sequestered from RA signaling and do not initiate meiosis until after birth.
进入性腺后不久,生殖细胞在胎儿性腺中获得减数分裂起始和性分化的能力。生殖细胞是启动减数分裂还是继续进行有丝分裂细胞周期取决于它们的性腺环境,而不是它们的性染色体构成(在 [20] 中综述)。胎儿卵巢中的生殖细胞暴露于 RA 并启动减数分裂,而胎儿睾丸中的生殖细胞与 RA 信号分离,直到出生后才开始减数分裂。

2.1. Formation of the Gonad and Migration of PGCs to the Gonad
2.1. 性腺的形成和 PGC 向性腺的迁移

In mammals, both the ovary and testis derive from a common precursor structure, the bipotential gonad (Figure 1) [21]. The development of the bipotential gonad involves two simultaneously occurring processes. The coelomic epithelium develops into a thickened, multilayer structure, known as the genital ridge. This differentiation initiates at the coelomic epithelium’s anterior end and extends posteriorly [3,21]. In mice, the development of the bipotential gonad begins at around E10.0 and continues until E11.5–E12.0 [1,2,3]. Thereafter, the gonad’s somatic cells undergo sexual differentiation [1,2,3].
在哺乳动物中,卵巢和睾丸都来源于一个共同的前体结构,即双电位性腺(图 1)[21]。双电位性腺的发育涉及两个同时发生的过程。体腔上皮发育成增厚的多层结构,称为生殖器嵴。这种分化始于体腔上皮的前端,并向后延伸[3,21]。在小鼠中,双电位性腺的发育从 E10.0 左右开始,一直持续到 E11.5-E12.0 [1,2,3]。此后,性腺的体细胞发生性分化[1,2,3]。
Figure 1. Anterior-to-posterior wave of Dazl and Stra8 expression from E10.5 to E14.5 in mouse fetal gonads. Germ cells are shown in circles, with cells expressing Dazl shown in orange, and cells expressing Stra8 and Dazl shown in blue. After gonadal colonization, germ cells continue to proliferate until E13.5 [22]. In the fetal mouse testis, germ cells become enclosed by somatic cells, with testis cords formed between E12.5 to E14.0 [14,23].
图 1.小鼠胎儿性腺中 DazlStra8 的前后波表达从 E10.5 到 E14.5。生殖细胞以圆圈显示,表达 Dazl 的细胞以橙色显示,表达 Stra8Dazl 的细胞以蓝色显示。性腺定植后,生殖细胞继续增殖,直到 E13.5 [22]。在胎鼠睾丸中,生殖细胞被体细胞包围,睾丸索形成于E12.5至E14.0之间[14,23]。
Meanwhile, PGCs, the precursors of sperm and eggs, are induced early in embryogenesis, and later migrate to the developing genital ridge [22]. Throughout their migration, PGCs maintain a transcriptional program of developmentally uncommitted cells, marked by the expression of both naïve and general pluripotency factors [24,25,26]. Upon colonization of the nascent gonad, human and mouse PGCs induce a set of germ cell factors, including evolutionarily conserved markers of germ granules [24]. After their arrival in the gonad, PGCs subsequently down-regulate the expression of pluripotency factors, and lose the capacity to give rise to pluripotent cell lines (known an embryonic germ [EG] cells) and teratomas, a tumor arising from pluripotent cells [24,27,28]. This transition, broadly conserved among vertebrates, serves to restrict the developmental potential of the mammalian germ line, a process termed germ cell determination [24].
同时,PGCs是精子和卵子的前体,在胚胎发生的早期被诱导,然后迁移到发育中的生殖器脊[22]。在整个迁移过程中,PGC 维持着发育未定型细胞的转录程序,其特征是幼稚和一般多能性因子的表达 [24,25,26]。在新生性腺定植后,人和小鼠 PGC 诱导一组生殖细胞因子,包括生殖颗粒的进化保守标志物 [24]。到达性腺后,PGC随后下调多能性因子的表达,并失去产生多能细胞系(称为胚胎胚芽[EG]细胞)和畸胎瘤(一种由多能细胞引起的肿瘤)的能力[24,27,28]。这种转变在脊椎动物中广泛保守,有助于限制哺乳动物生殖系的发育潜力,这一过程称为生殖细胞决定[24]。

2.2. Initiation of Gametogenesis and Meiotic Entry
2.2. 配子发生和减数分裂进入

Once determined, germ cells are poised to initiate meiosis, as well as undertake male or female differentiation [29,30]. The transition of PGCs to committed germ cells represents a critical transformation of the germ line to a sexually competent state [31], and is induced by extrinsic signals from the genital ridge [32]. One of the genes induced at PGC colonization in mice and humans is Dazl [24], which encodes an evolutionarily conserved and germ-cell-specific RNA-binding protein (Figure 2) [33]. In Dazl-null mouse embryos, PGCs arrive at the gonad, but fail to restrict their developmental potential; instead, these cells remain proliferative, continue to express pluripotency factors, retain the capacity for the derivation of pluripotent EG cells until at least E15.5, and fail to initiate meiosis or embark upon spermatogenesis or oogenesis in the fetal testis or ovary, respectively [24,31,34,35]. Consistent with the failure to restrict germline potential, Dazl-deficient mice and pigs develop spontaneous teratomas at an elevated frequency [24]. Thus, Dazl is necessary for the germ line to undertake a restriction of potential, and for the competence to undertake gametogenesis, defined as the capacity to initiate meiosis and sexual differentiation [31].
一旦确定,生殖细胞就会开始减数分裂,并进行雄性或雌性分化[29,30]。PGC 向定型生殖细胞的转变代表了生殖系向性功能状态的关键转变 [31],并且是由来自生殖器嵴的外源信号诱导的 [32]。在小鼠和人类中PGC定植时诱导的基因之一是Dazl [24],它编码一种进化上保守的生殖细胞特异性RNA结合蛋白(图2)[33]。在 Dazl 缺失小鼠胚胎中,PGC 到达性腺,但未能限制其发育潜力;相反,这些细胞保持增殖,继续表达多能因子,保留多能EG细胞的衍生能力,直到至少E15.5,并且无法分别在胎儿睾丸或卵巢中启动减数分裂或开始精子发生或卵子发生[24,31,34,35]。与未能限制种系潜力的情况一致,Dazl 缺陷小鼠和猪发生自发性畸胎瘤的频率较高 [24]。因此,Dazl 对于种系进行潜力限制以及进行配子发生的能力是必需的,配子发生定义为启动减数分裂和性分化的能力 [31]。
Figure 2. Diagram of germ cell development in mouse fetal gonads of both sexes. Red box: female gonad (ovary). Blue box: male gonad (testis). DAZL, STRA8, REC8, and NANOS2 are expressed in germ cells. ALDH1A1 and CYP26B1 are expressed in fetal gonads. ALDH1A2 and ALDH1A3 are expressed outside the gonads.
图 2.两性小鼠胎儿性腺的生殖细胞发育图。红框:女性性腺(卵巢)。蓝色框:雄性性腺(睾丸)。DAZL、STRA8、REC8 和 NANOS2 在生殖细胞中表达。ALDH1A1 和 CYP26B1 在胎儿性腺中表达。ALDH1A2 和 ALDH1A3 在性腺外表达。
On expression of DAZL, germ cells acquire the competence to interpret RA as a meiosis-inducing signal (Figure 2) [7,8,35]. RA induces germ cells to express both Stra8 (Stimulated by Retinoic acid gene 8), a gene required for meiotic initiation [36], and Rec8, a gene required for meiotic progression [37,38] (Figure 1 and Figure 2) [39,40]. These two factors are independently activated by RA (Figure 2) [39,41] and precede the expression of other meiotic markers, such as Dmc1, Sycp3, and the phosphorylation of histone H2AX (γH2AX), which is a marker of meiotic double strand breaks [26,40,42,43].
DAZL 表达时,生殖细胞获得将 RA 解释为减数分裂诱导信号的能力(图 2)[7,8,35]。RA诱导生殖细胞表达减数分裂起始所需的基因Stra8受视黄酸基因8刺激[36]和减数分裂进展所需的基因Rec8[37,38](图1图2)[39,40]。这两个因子被RA独立激活(图2)[39,41],并先于其他减数分裂标志物的表达,如Dmc1Sycp3和组蛋白H2AX(γH2AX)的磷酸化,后者是减数分裂双链断裂的标志物[26,40,42,43]。
Like the preceding differentiation of the somatic cells, many aspects of germline development occur in an anterior-to-posterior (A-P) wave along the length of the gonad [26,32,40,42,44]. At E11.5, newly arrived PGCs show a gradient of Dazl expression, which is highest in the anterior portion of the gonad and low or absent in the posterior portion (Figure 1) [32].
与前面的体细胞分化一样,种系发育的许多方面都发生在沿性腺长度的前后 (A-P) 波中 [26,32,40,42,44]。在 E11.5 处,新到达的 PGC 显示出 Dazl 表达的梯度,该梯度在性腺的前部最高,在后部较低或不存在(图 1)[32]。

2.3. Stra8 and Its Inducer, RA, Regulate Meiotic Initiation in the Fetal Ovary
2.3. Stra8 及其诱导剂 RA 调节胎儿卵巢的减数分裂启动

Stra8 is highly expressed in germ cells of both sexes at meiotic initiation, before quickly turning off early in meiosis [18,36,40]. Stra8 expression in ovarian germ cells begins at E12.5 and progresses in a subsequent A-P wave, such that the expression of Stra8 and other meiotic markers is heterogeneous across the population of germ cells (Figure 1) [26,39,40]. In the fetal ovary, Stra8 is first detected within one day prior to when the characteristically condensed chromatin of meiotic germ cells can be observed (Figure 1 and Figure 2) [40]. In mice of the C57BL/6 genetic background, Stra8-null ovarian germ cells do not undergo meiotic DNA replication [36], nor do they robustly express meiotic factors or begin the chromosomal events of meiotic prophase I [36,41]; thus, Stra8 is necessary for meiotic initiation in mice. STRA8 is a transcriptional activator that binds to the promoters and enhances the expression of thousands of genes, including meiotic prophase I genes, G1-S cell-cycle genes, and factors that specifically inhibit the mitotic program [45]. In fetal testes, male germ cells do not express Stra8 (Figure 1 and Figure 2) [40]. Instead, Stra8 is first expressed much later in germ cells of postnatal testes, when they undergo differentiation [46,47,48].
Stra8 在减数分裂开始时在两性生殖细胞中都高度表达,然后在减数分裂早期迅速关闭 [18,36,40]。Stra8在卵巢生殖细胞中的表达从E12.5开始,并在随后的A-P波中进展,因此Stra8和其他减数分裂标志物的表达在生殖细胞群中是异质性的(图1)[26,39,40]。在胎儿卵巢中,Stra8 在可以观察到减数分裂生殖细胞特征性浓缩染色质之前的 1 天内首次检测到(图 1图 2)[40]。在 C57BL/6 遗传背景的小鼠中,Stra8 缺失的卵巢生殖细胞不进行减数分裂 DNA 复制 [36],它们也不强烈表达减数分裂因子或开始减数分裂前期 I 的染色体事件 [36,41];因此,Stra8 是小鼠减数分裂起始所必需的。STRA8 是一种转录激活剂,可与启动子结合并增强数千个基因的表达,包括减数分裂前期 I 基因、G1-S 细胞周期基因和特异性抑制有丝分裂程序的因子 [45]。在胎儿睾丸中,雄性生殖细胞不表达Stra8图1图2)[40]。相反,Stra8 在出生后睾丸的生殖细胞中首次表达的时间要晚得多,当它们发生分化时 [46,47,48]。
A potential link between RA and meiotic initiation was initially provided by in vivo studies of the Stra8 gene [36,40], which was first identified as an RA-inducible gene in embryonal carcinoma cells and embryonic stem cells in vitro [46]. In fetal ovaries, all-trans RA robustly induces Stra8 expression and thereby meiotic initiation (Figure 2) [7,8]. Exogenous all-trans RA is sufficient to induce ectopic Stra8 expression, and for the precocious initiation of meiosis in fetal testes [7,8]. Later work provided direct evidence for RA’s role in meiotic initiation—in the ovaries of vitamin A-deficient rat embryos, Stra8 is not robustly activated, and germ cells fail to enter meiosis [49]. Thus, RA can induce meiotic initiation in both female and male germ cells of the fetal gonad.
RA与减数分裂起始之间的潜在联系最初是由Stra8基因的体内研究提供的[36,40],该基因最初在体外胚胎癌细胞和胚胎干细胞中被鉴定为RA诱导基因[46]。在胎儿卵巢中,全式 RA 强烈诱导 Stra8 表达,从而诱导减数分裂起始(图 2)[7,8]。外源性全式 RA 足以诱导异位 Stra8 表达,并导致胎儿睾丸减数分裂早熟 [7,8]。后来的研究为 RA 在减数分裂起始中的作用提供了直接证据——在维生素 A 缺乏的大鼠胚胎的卵巢中,Stra8 没有被强烈激活,生殖细胞无法进入减数分裂 [49]。因此,RA 可以在胎儿性腺的雌性和雄性生殖细胞中诱导减数分裂起始。
Two families of nuclear hormone receptors, known as RA receptors (RARs) and retinoid X receptors (RXRs), bind RA. RARs bind both all-trans and 9-cis RA stereoisoforms, while RXRs bind only 9-cis RA [50]. RXRs can also bind other ligands that are not derived from RA [51] (reviewed in [52,53]), but it is unclear whether these ligands contribute to meiotic initiation. RARs and RXRs interact to form heterodimers that bind to RA response elements (RAREs) in the regulatory regions of target genes [54]. RXRs can also heterodimerize with other nuclear hormone receptors (reviewed in [55]), but whether these interactions promote meiotic initiation is not yet known. RARs and RXRs each have three isotypes (RARα, RARβ, and RARγ, and RXRα, RXRβ, and RXRγ), and each exhibits overlapping expression and functional redundancy in many tissues (reviewed in [54,56,57]). Both RAR and RXR isotypes are expressed in the gonads of each sex [8,58,59,60,61]. In embryonic ovaries, RARs are readily detected in germ cells but are expressed at very low levels, if at all, in somatic cells [8,58,62], while RXRs are found in both somatic and germ cells [8,62]. The promoter of the Stra8 gene contains two putative RAREs, suggesting that RA may directly up-regulate Stra8 transcription by binding to RAR/RXR heterodimers engaged at the Stra8 promoter [46,63]. Indeed, antagonists of the RARs diminish or block Stra8 expression, while exogenous RA induces Stra8 expression in the fetal ovary [7,8].
两个核激素受体家族,称为 RA 受体 (RAR) 和类视黄醇 X 受体 (RXR),结合 RA。RAR 可结合全式和 9-顺式 RA 立体异构体,而 RXR 仅结合 9-顺式 RA [50]。RXR还可以结合非RA来源的其他配体[51](在[52,53]中已论述),但尚不清楚这些配体是否有助于减数分裂起始。RAR 和 RXR 相互作用形成异二聚体,这些异二聚体与靶基因调控区域中的 RA 反应元件 (RARE) 结合 [54]。RXR 也可以与其他核激素受体异二聚化(在 [55] 中综述),但这些相互作用是否促进减数分裂起始尚不清楚。RARs 和 RXRs 各有 3 种同种型(RARα、RARβ 和 RARγ,以及 RXRα、RXRβ 和 RXRγ),并且每种同种型在许多组织中都表现出重叠的表达和功能冗余(在 [54,56,57] 中已论述)。RAR 和 RXR 同种型在每种性别的性腺中都有表达 [8,58,59,60,61]。在胚胎卵巢中,RARs很容易在生殖细胞中检测到,但在体细胞中的表达水平非常低[8,58\u201262],而RXRs在体细胞和生殖细胞中都存在[8,62]。Stra8 基因的启动子包含两个推定的 RARE,表明 RA 可能通过与 Stra8 启动子啮合的 RAR/RXR 异二聚体结合来直接上调 Stra8 转录 [46,63]。事实上,RARs 的拮抗剂会减少或阻断 Stra8 的表达,而外源性 RA 会诱导胎儿卵巢中 Stra8 的表达 [7,8]。

2.4. Source of RA in the Fetal Ovary
2.4. 胎卵巢 RA 的来源

RA originating from both the somatic cells of the fetal ovary and mesonephros likely contribute to meiotic initiation (Figure 2) [8,9]. Initial studies identified the mesonephros as a robust source of RA, as these cells strongly expressed a lacZ reporter transgene under the control of an RARE [8]. Weaker RARE-lacZ signal was detected in the fetal gonad, with the strongest gonadal signal detected at the anterior end [8]. The mesonephros expresses two RA-synthesizing enzymes (Figure 2), aldehyde dehydrogenase 1A2 (Aldh1a2) [8] and Aldh1a3 [64]. Upon deletion of Aldh1a2 or both Aldh1a2 and Aldh1a3, the mesonephros fails to produce RA, as evidenced by the loss of RARE-lacZ signal in transgenic mice [65]. At the same time, the ovarian germ cells from these mutant embryos express Stra8 and initiate meiosis [65]. Therefore, mesonephros-derived RA is not strictly required for meiotic initiation.
源自胎儿卵巢体细胞和中肾的 RA 可能有助于减数分裂的启动(图 2)[8,9]。初步研究确定中肾是 RA 的可靠来源,因为这些细胞在 RARE 的控制下强烈表达 lacZ 报告基因转基因 [8]。在胎儿性腺中检测到较弱的 RARE-lacZ 信号,在前端检测到最强的性腺信号 [8]。中肾表达两种 RA 合成酶(图 2),醛脱氢酶 1A2Aldh1a2) [8] 和 Aldh1a3 [64]。当 Aldh1a2Aldh1a2Aldh1a3 缺失时,中肾无法产生 RA,转基因小鼠中 RARE-lacZ 信号的丢失证明了这一点 [65]。同时,来自这些突变胚胎的卵巢生殖细胞表达 Stra8 并启动减数分裂 [65]。因此,中肾衍生的 RA 并不是减数分裂起始的严格要求。
Based on these findings, some have proposed that RA itself is not required for meiotic initiation in the ovary [65]. However, subsequent work demonstrated that germ cells from cultured fetal ovaries initiate meiosis in the absence of the mesonephros, suggesting that an alternative source of RA—such as the fetal ovary—is sufficient for meiotic initiation [66]. Additional studies indicated that the somatic cells of the fetal gonad express Aldh1a1 and therefore produce RA (Figure 2) [9,66,67]. Further, genetic deletion of Aldh1a1 decreases RA levels in the fetal ovary [9]. While Aldh1a1-deficient fetal ovaries initially exhibit reduced expression of Stra8 and other genes that are usually upregulated at meiotic initiation, these meiotic factors are expressed at similar levels one day later, suggesting that RA derived from the mesonephros allows the germ cells to initiate meiosis and overcome the earlier delay [9]. Consistent with this recovery, Aldh1a1-null female mice are fertile [68]. Therefore, RA derived from the fetal ovary via Aldh1a1 regulates the timing of meiotic initiation, but is not strictly required. At the same time, Aldh1a1 provides sufficient RA to initiate meiosis in the ovaries of Aldh1a2-null; Aldh1a3-null embryos.
基于这些发现,一些人提出 RA 本身并不是卵巢减数分裂起始所必需的 [65]。然而,随后的工作表明,来自培养的胎儿卵巢的生殖细胞在没有中肾的情况下启动减数分裂,这表明 RA 的替代来源(例如胎儿卵巢)足以进行减数分裂启动 [66]。其他研究表明,胎儿性腺的体细胞表达 Aldh1a1,因此产生 RA(图 2)[9,66,67]。此外,Aldh1a1 的基因缺失会降低胎儿卵巢中的 RA 水平 [9]。虽然 Aldh1a1 缺陷的胎儿卵巢最初表现出 Stra8 和其他基因的表达降低,这些基因通常在减数分裂开始时上调,但这些减数分裂因子在一天后以相似的水平表达,表明来源于中肾的 RA 允许生殖细胞启动减数分裂并克服早期的延迟 [9]。与这种恢复一致,Aldh1a1 缺失的雌性小鼠具有生育能力 [68]。因此,通过 Aldh1a1 从胎儿卵巢衍生的 RA 调节减数分裂开始的时间,但并非严格要求。同时,Aldh1a1 提供足够的 RA 以启动 Aldh1a2-null 卵巢中的减数分裂;Aldh1a3 缺失胚胎。
That Aldh1a1 is redundant for meiotic initiation may be accounted for by its inverse expression in response to RA levels. In fetal testes lacking Cyp26b1, endogenous RA levels are elevated, and Aldh1a1 expression is greatly reduced, suggesting a negative feedback loop between RA signaling and Aldh1a1 expression [9]. Therefore, the elimination of mesonephros-derived RA by deletion of Aldh1a2 and Aldh1a3 may cause an increase in Aldh1a1 expression in the gonad, raising RA levels in the fetal ovary [9]. In the embryonic ovary, RA produced by both the mesonephros and somatic gonad likely contributes to meiotic initiation.
Aldh1a1 对于减数分裂起始是多余的,这可能是由于它响应 RA 水平的逆表达。在缺乏 Cyp26b1 的胎儿睾丸中,内源性 RA 水平升高,Aldh1a1 表达大大降低,表明 RA 信号转导和 Aldh1a1 表达之间存在负反馈回路 [9]。因此,通过缺失 Aldh1a2Aldh1a3 消除中肾源性 RA 可能导致性腺中 Aldh1a1 表达增加,从而提高胎儿卵巢中的 RA 水平 [9]。在胚胎卵巢中,中肾和体细胞性腺产生的 RA 可能有助于减数分裂的启动。
Early studies of RA activity proposed that RA diffuses through the fetal gonad in an A-P manner to produce an A-P wave of meiotic initiation (Figure 1 and Figure 2) (reviewed in [69,70]). While the mesonephros is attached to the gonad along its dorsal length, only the anterior mesonephric tubules are open and directly connected to the gonad (Figure 1) [71,72]. Thus, RA may diffuse from the mesonephros into the gonad via this anterior connection [8] (reviewed in [69]). Alternatively, some RA-producing cells may migrate from the anterior mesonephros into the anterior gonad (reviewed in [69]). Both scenarios could establish an A-P gradient that drives the observed wave of meiotic initiation. Consistent with this model, the RARE-lacZ reporter is detected in the fetal ovary in an A-P manner [8,9].
对 RA 活性的早期研究表明,RA 以 A-P 方式通过胎儿性腺扩散,产生减数分裂起始的 A-P 波(图 1图 2)(在 [69,70] 中综述)。虽然中肾沿其背长附着在性腺上,但只有前中肾小管是开放的,并与性腺直接相连(图1)[71,72]。因此,RA 可能通过这个前连接从中肾扩散到性腺中 [8](在 [69] 中已论述)。或者,一些产生 RA 的细胞可能会从前中肾迁移到前性腺(在 [69] 中综述)。这两种情况都可以建立一个 A-P 梯度,驱动观察到的减数分裂起始波。与该模型一致,在胎儿卵巢中以A-P方式检测到RARE-lacZ报告基因[8,9]。
An A-P wave of Dazl expression precedes, and may also contribute to, the subsequent wave of meiotic initiation (Figure 1) [32]. On Dazl expression, germ cells acquire the ability to interpret RA as a meiosis-inducing factor [35] in an A-P manner (Figure 1) [32]. This wave of intrinsic germ cell competence may reinforce an RA gradient in inducing meiosis along the gonad. Alternatively, the A-P wave of intrinsic germ cell competence may drive the subsequent wave of meiotic initiation, independent of any differences in the local concentration of RA along the length of the gonad. Regardless, RA can induce Dazl expression in cultured PGC-like cells [73], which suggests an additional instructive role for RA in the development of germ cells in both the XX and XY-bearing cells, days prior to meiotic initiation.
Dazl 表达的 A-P 波先于随后的减数分裂起始波,也可能有助于随后的减数分裂起始波(图 1)[32]。在 Dazl 表达时,生殖细胞获得了以 A-P 方式将 RA 解释为减数分裂诱导因子的能力 [35](图 1)[32]。这种内在生殖细胞能力的波动可能会加强 RA 梯度,以诱导沿性腺减数分裂。或者,内在生殖细胞能力的 A-P 波可以驱动随后的减数分裂起始波,而与沿性腺长度的 RA 局部浓度的任何差异无关。无论如何,RA可以在培养的PGC样细胞中诱导Dazl表达[73],这表明RA在减数分裂开始前几天在携带XX和XY的细胞中生殖细胞的发育中具有额外的指导作用。

2.5. Prevention of Meiotic Initiation in the Fetal Testis
2.5. 预防胎儿睾丸减数分裂启动

In fetal testes, CYP26B1 degrades RA, thereby precluding the induction of Stra8, and preventing the initiation of meiosis (Figure 2) [7,8]. Cyp26b1 is expressed in somatic cells of the developing testis (seminiferous) cords [7,8,74,75]. In Cyp26b1-deficient embryos, germ cells in the fetal testis express ectopic Stra8 and initiate meiosis [8,76]. Thus, CYP26B1-expressing cells form a catabolic barrier that prevents RA, generated outside of the cords, from reaching the germ cells located within. The expression level of Cyp26b1 in mouse fetal testes is maintained until E13.5, and reduced gradually thereafter [77]. The subsequent reduction of Cyp26b1 may expose male germ cells to some RA, but male germ cells avoid a direct response, in part, through Nanos2, which prevents meiotic initiation in the fetal testis [77,78,79] (reviewed in [70,80,81]). The expression of Nanos2, which encodes a germ cell-specific RNA binding protein [82], is up-regulated from E13.5 onward and is restricted to the male germline [77,83]. In Nanos2-null embryos, male germ cells express low levels of Stra8 and initiate ectopic meiosis at E14.5 [77], indicating that Nanos2 operates subsequent to RA catabolism by Cyp26b1 to prevent cells from initiating meiosis. The authors also reported that Nanos2 inhibits meiosis, in part, by destabilizing Dazl and other down-stream targets (Figure 2) [79]. Thus, Nanos2 is a cell-intrinsic factor that prevents the male germline from interpreting RA as a meiosis-inducing factor.
在胎儿睾丸中,CYP26B1降解 RA,从而排除 Stra8 的诱导,并阻止减数分裂的开始(图 2)[7,8]。Cyp26b1 在发育中的睾丸(生精)索的体细胞中表达 [7,8,74,75]。Cyp26b1 缺陷的胚胎中,胎儿睾丸中的生殖细胞表达异位 Stra8 并启动减数分裂 [8,76]。因此,表达 CYP26B1 的细胞形成分解代谢屏障,阻止在脊髓外产生的 RA 到达位于内部的生殖细胞。小鼠胎儿睾丸中 Cyp26b1 的表达水平维持到 E13.5,此后逐渐降低 [77]。随后 Cyp26b1 的减少可能会使雄性生殖细胞暴露于一些 RA,但雄性生殖细胞部分避免了通过 Nanos2 的直接反应,从而阻止了胎儿睾丸的减数分裂起始 [77,78,79](在 [70,80,81] 中已论述)。Nanos2编码生殖细胞特异性RNA结合蛋白[82],其表达从E13.5开始上调,仅限于雄性种系[77,83]。Nanos2 缺失胚胎中,雄性生殖细胞表达低水平的 Stra8 并在 E14.5 处启动异位减数分裂 [77],表明 Nanos2Cyp26b1 的 RA 分解代谢之后发挥作用,以防止细胞启动减数分裂。作者还报道,Nanos2 在一定程度上通过破坏 Dazl 和其他下游靶标的稳定性来抑制减数分裂(图 2)[79]。 因此,Nanos2 是一种细胞内因子,可防止雄性种系将 RA 解释为减数分裂诱导因子。

2.6. A Role for RA in the Ovary after Birth
2.6. RA 在出生后卵巢中的作用

After meiotic initiation, ovarian germ cells enter an extended meiotic prophase I, and begin differentiation as oocytes [36]. In mice, oocytes that progress through meiotic prophase I will arrest at the diplotene stage, also known as dictyate or germinal vesicle (GV) stage, around birth (reviewed in [84,85,86]). Shortly after birth, oocytes grow and differentiate independent of the chromosomal events of meiosis [87]. Meanwhile, oocytes organize the supporting somatic cells, called granulosa cells, to form follicles [88], which later undertake ovulation in response to hormonal stimulation. During and after puberty, groups of follicles will grow in size through both granulosa cell proliferation and the growth of the oocyte, which remain arrested at the GV stage (reviewed in [86]). Around the time of ovulation, full-grown GV stage oocytes resume meiosis, break down the nuclear envelope (GV breakdown), undergo meiotic progression, and arrest again at meiotic metaphase II (MII) until fertilization; the process from GV to MII stage is referred to as oocyte maturation, which is promoted by granulosa cells (reviewed in [84,85]).
减数分裂开始后,卵巢生殖细胞进入扩展的减数分裂前期I,并开始分化为卵母细胞[36]。在小鼠中,通过减数分裂前期I的卵母细胞将在出生前后的diplotene阶段停滞,也称为dictyate或生发囊泡(GV)阶段([84,85,86])出生后不久,卵母细胞的生长和分化与减数分裂的染色体事件无关 [87]。同时,卵母细胞组织支持体细胞(称为颗粒细胞)形成卵泡[88],卵泡随后在激素刺激下进行排卵。在青春期和青春期之后,卵泡群会通过颗粒细胞增殖和卵母细胞的生长而变大,卵母细胞在 GV 阶段仍然停滞不前(在 [86] 中综述)。在排卵前后,完全生长的 GV 期卵母细胞恢复减数分裂,打破核膜(GV 崩溃),经历减数分裂进展,并在减数分裂中期 II (MII) 再次停滞直至受精;从 GV 到 MII 阶段的过程称为卵母细胞成熟,由颗粒细胞促进(在 [84,85] 中已论述)。
Recent in vitro studies have proposed that both all-trans and 9-cis RA can act on granulosa cells to improve oocyte maturation in several mammals, including cows [89,90,91,92,93], goats [94], pigs [95], rats [96], and mice [97,98] (reviewed in [99,100]). RARs and RXRs are expressed in granulosa cells surrounding full-grown oocytes [96,101,102]. Supplementation of culture medium with all-trans or 9-cis RA induces granulosa cells to express genes that regulate differentiation and prevent apoptosis [90,92,93,94,103,104] (reviewed in [99]), suggesting that RA acts on granulosa cells to prevent their aberrant differentiation state and apoptosis. In vivo, RARE-lacZ signal is detected in granulosa cells of mouse ovarian follicles at 3 weeks of age, and increased after injection of a gonadotropic hormone [102], supporting a role for RA on these cells. Further in vivo studies are needed to determine whether RA is required by granulosa cells to support oocyte maturation in the ovary.
最近的体外研究表明,全式和9-顺式RA都可以作用于颗粒细胞,以改善几种哺乳动物的卵母细胞成熟,包括奶牛[89,90,91,92,93]、山羊[94]、猪[95]、大鼠[96]和小鼠[97,98]([99,100]综述)。RAR和RXR在成熟卵母细胞周围的颗粒细胞中表达[96,101,102]。在培养基中补充全式或 9-顺式 RA 可诱导颗粒细胞表达调节分化和防止细胞凋亡的基因 [90,92,93,94,103,104](在 [99] 中已论述),表明 RA 作用于颗粒细胞以防止其异常分化状态和细胞凋亡。在体内,在 3 周龄小鼠卵巢卵泡的颗粒细胞中检测到 RARE-lacZ 信号,并在注射促性腺激素后增加 [102],支持 RA 在这些细胞上的作用。需要进一步的体内研究来确定颗粒细胞是否需要 RA 来支持卵巢中的卵母细胞成熟。

3. Development of Male Germ Cells after Birth
3. 出生后雄性生殖细胞的发育

After birth, male germ cells differentiate into spermatogonia and initiate spermatogenesis, a process in which spermatogonial stem cells ultimately give rise to millions of haploid spermatozoa per day. Throughout spermatogenesis, several transitions occur in a strictly coordinated manner, including meiotic initiation, which is induced by periodic RA signaling, ensuring that spermatozoa are produced at a constant rate throughout reproductive life in males.
出生后,雄性生殖细胞分化成精原细胞并启动精子发生,在这个过程中,精原干细胞最终每天产生数百万个单倍体精子。在整个精子发生过程中,几个转变以严格协调的方式发生,包括由周期性 RA 信号诱导的减数分裂起始,确保精子在雄性的整个生殖生活中以恒定的速度产生。

3.1. Organization of Spermatogenesis in the Postnatal and Adult Testis
3.1. 出生后和成人睾丸精子发生的组织

In the fetal mouse testis, PGCs are enclosed by somatic cells as testis cords are formed between E12.5 to E14.0 (Figure 1) [14,23]. The germ cells present within the testis cords differ morphologically from migratory PGCs, and are called gonocytes [14,15]. Shortly after birth, the gonocytes, which are arrested in the G0/G1 phase [4,5], resume proliferation and migrate to the basement of the cords to give rise to type A spermatogonia (Figure 3) [14,15,105].
在胎鼠睾丸中,PGC 被体细胞包围,因为睾丸索在 E12.5 至 E14.0 之间形成(图 1)[14,23]。睾丸索内的生殖细胞在形态上与迁移性PGC不同,称为性腺细胞[14,15]。出生后不久,停滞在 G0/G1 期的淋病细胞 [4,5] 恢复增殖并迁移到脊髓基底,产生 A 型精原细胞(图 3)[14,15,105]。
Figure 3. Structure of the mouse testis comprising seminiferous tubules. In any given tubule cross-section, one observes germ cells at different steps of their development into elongated spermatids. These germ cell types are concentrically layered; undifferentiated spermatogonia lie on the basal lamina of the tubule, and germ cells move toward the tubule lumen as they differentiate [106]. Germ cell differentiation is precisely timed; hence, particular steps of development are always found together in close physical proximity. Blue line indicates the orientation of testis cross-sections. A representative tubule cross-section in stage VII–VIII, stained with hematoxylin and periodic acid-Schiff (He-PAS), is shown with grayscale version. Star: Sertoli cell nucleus. White arrowhead: type spermatogonium. Dots: preleptotene (red) spermatocytes, pachytene spermatocytes (yellow), and step 7–8 round spermatids (green). Brown area: elongated spermatids. Scale bar = 30 μm.
图 3.小鼠睾丸的结构由生精小管组成。在任何给定的小管横截面中,人们可以观察到生殖细胞发育成细长精子细胞的不同步骤。这些生殖细胞类型是同心分层的;未分化的精原细胞位于肾小管的基底层,生殖细胞在分化时向肾小管腔移动[106]。生殖细胞分化的时间精确;因此,特定的发展步骤总是在物理上非常接近的情况下一起被发现。蓝线表示睾丸横截面的方向。VII-VIII 期的代表性小管横截面,用苏木精和过碘酸希夫 (He-PAS) 染色,以灰度版本显示。星号:支持细胞核。白色箭头:类型 spermatogonium。点:preleptotene(红色)精母细胞、厚层精母细胞(黄色)和第 7-8 步圆形精母细胞(绿色)。棕色区域:细长的精子细胞。比例尺 = 30 μm。
In mice, spermatogenesis begins with undifferentiated type A spermatogonia, which include the stem cells [107,108,109,110] (reviewed in [111]). Individual spermatogonial cells, known as A single (As) spermatogonia, have traditionally been considered to encompass spermatogonial stem cells (Figure 4) [107,108,112]. Some of the As spermatogonia divide into paired A (Apr) spermatogonia, which are connected by an intercellular bridges. The Apr spermatogonia subsequently divide further into extended chains of 4, 8, or 16 cells, called Aaligned (Aal) spermatogonia. As, Apr, and Aal spermatogonia are referred to as undifferentiated spermatogonia (Figure 4) (reviewed in [113]).
在小鼠中,精子发生从未分化的 A 型精原细胞开始,其中包括干细胞 [107,108,109,110](在 [111] 中已论述)。单个精原细胞,称为单个 (As) 精原细胞,传统上被认为包含精原干细胞(图 4)[107,108,112]。一些 As 精原细胞分裂成对的 A (Apr) 精原细胞,它们通过细胞间桥连接。Apr 精原细胞随后进一步分裂成 4、8 或 16 个细胞的延长链,称为 A对齐 (Aal) 精原细胞。As、Apr 和 Aal 精原细胞被称为未分化精原细胞(图 4)(在 [113] 中综述)。
Figure 4. Multiplication of undifferentiated spermatogonia and spermatogonial differentiation. Upon division, the Asingle (As) spermatogonia can self-renew and produce two new singles or the daughter cells, Apaired (Apr) spermatogonia, remain connected by an intercellular bridge. The Apr spermatogonia subsequently divide further into chains of 4, 8, or 16 cells, called Aaligned (Aal) spermatogonia that undergo spermatogonial differentiation (purple) in response to RA. As, Apr, and Aal spermatogonia are referred to as undifferentiated spermatogonia. After the spermatogonial differentiation, Aal spermatogonia transit into A1 differentiating spermatogonia without a mitotic division [114]. Expression patterns of PLZF, RARγ, STRA8, SALL4, and KIT are indicated as solid lines.
图 4.未分化精原细胞的增殖和精原分化。分裂后,A单个 (As) 精原细胞可以自我更新并产生两个新的单精子,或者子细胞 A (Apr) 精原细胞通过细胞间桥保持连接。Apr 精原细胞随后进一步分裂成 4、8 或 16 个细胞的链,称为 A对齐 (Aal) 精原细胞,它们响应 RA 而经历精原分化(紫色)。As、Apr 和 Aal 精原细胞被称为未分化精原细胞。精原细胞分化后,Aal 精原细胞转运到 A1 分化的精原细胞,没有有丝分裂 [114]。PLZF 、 RARγ 、 STRA8 、 SALL4 和 KIT 的表达模式用实线表示。
Undifferentiated spermatogonia periodically commit to differentiation, in the form of an Aal-to-A1 transition, to become differentiating spermatogonia, which encompass A1, A2, A3, A4, intermediate and B spermatogonia (Figure 4 and Figure 5) (reviewed in [114,115]). During differentiation, spermatogonia lose the capacity for self-renewal [116], accelerate their cell cycle [117], and undertake six mitotic divisions in mice [118]. Germ cells then differentiate to spermatocytes and undergo meiotic initiation (Figure 5) [18,36]. DNA replication and two cell divisions follow, resulting in the formation of haploid, round spermatids, which elongate their nucleus and cytoplasm to become elongated spermatids. Finally, these spermatids are released into the lumen of the seminiferous epithelium, whereupon they are referred to as spermatozoa (Figure 3 and Figure 5) (reviewed in [119]). These layered generations of germ cells are embedded in and supported by somatic Sertoli cells that supply factors essential for spermatogenesis (Figure 3) (reviewed in [120,121]).
未分化的精原细胞周期性地以 A al-to-A1 转变的形式进行分化,成为分化精原细胞,包括 A1、A2、A3、A4、中间和 B 精原细胞(图 4图 5)(在 [114,115] 中综述)。在分化过程中,精原细胞失去自我更新的能力[116],加速其细胞周期[117],并在小鼠中进行六次有丝分裂[118]。然后,生殖细胞分化为精母细胞并经历减数分裂起始(图 5)[18,36]。随后进行 DNA 复制和两次细胞分裂,形成单倍体圆形精子细胞,将其细胞核和细胞质拉长,成为细长的精子细胞。最后,这些精子细胞被释放到生精上皮的腔中,因此它们被称为精子(图 3图 5)(在 [119] 中综述)。这些分层的生殖细胞代嵌入体细胞支持细胞中并得到其支持,这些细胞提供精子发生所必需的因子(图 3)(在 [120,121] 中已论述)。
Figure 5. Diagram of mouse spermatogenesis. Oakberg [106] identified 12 distinct cellular associations, called seminiferous stages I–XII. It takes 8.6 days for a section of seminiferous tubule, and the germ cells contained within, to cycle through all 12 stages [122]. Four turns of this seminiferous cycle are required for a germ cell to progress from undifferentiated spermatogonium to spermatozoon. As, Apr, and Aal: Asingle, Apaired, and Aaligned spermatogonia. A1–A4: A1–A4 differentiating spermatogonia. In, and B: intermediate and type B spermatogonia. Pl, L, Z, P, D, and SC2: preleptotene, leptotene, zygotene, pachytene, diplotene, and secondary spermatocytes. Steps 1–16: steps in spermatid differentiation. Purple: germ cells undergoing spermatogonial differentiation; green: meiotic initiation; brown: initiation of spermatid elongation; gray: release of elongated spermatids. Black box: population of undifferentiated spermatogonia. Gray box: the leptotene spermatocytes undergoing migration of basal to luminal compartment [123]. Dark blue: stage with high RA concentration. Light blue line: STRA8 expression in the unperturbed testis. Dashed light blue line: STRA8 expression induced by RA injection in undifferentiated spermatogonia. (After RA injection, undifferentiated Aal spermatogonia in stages II–VI precociously express STRA8 [124]).
图 5.小鼠精子发生图。Oakberg [106] 确定了 12 个不同的细胞关联,称为生精阶段 I-XII。一段生精小管及其所含的生殖细胞需要 8.6 天才能循环完成所有 12 个阶段 [122]。生殖细胞从未分化的精原细胞发育到精子需要这个生精循环的四圈。As、Apr 和 Aal单个、A配对和 A对齐的精原细胞。A1-A 4:A1-A 4 分化精原细胞。In 和 B:中间和 B 型精原细胞。Pl、L、Z、P、D 和 SC2:preleptotene、leptotene、zygotene、pachytene、diplotene 和次级精母细胞。步骤 1-16:精子细胞分化的步骤。紫色:正在进行精原分化的生殖细胞;绿色:减数分裂起始;棕色:精子细胞伸长开始;灰色:细长的精子细胞释放。黑框:未分化精原细胞种群。灰色框:瘦素精母细胞从基底迁移到管腔隔室[123]。深蓝色:RA 浓度高的阶段。浅蓝线:STRA8 在未受干扰的睾丸中的表达。浅蓝色虚线:RA 注射在未分化精原细胞中诱导的 STRA8 表达。(RA 注射后,II-VI 期未分化的 Aal 精原细胞早熟表达 STRA8 [124])。
Within cross-sections of the seminiferous epithelium, stereotypical collections or associations of germ cells occur at various steps of differentiation (Figure 3 and Figure 5). The precise coordination of these steps is called the “cycle of the seminiferous epithelium” (or “seminiferous cycle”). In mice, the seminiferous cycle has been subdivided into 12 distinct cellular associations, known as seminiferous (epithelial) stages I to XII [106]. During spermatogenesis, four transitions direct key phases of germ cell development: (i) differentiation of spermatogonia, (ii) meiotic initiation, (iii) initiation of spermatid elongation, and (iv) the release of elongated spermatids into the lumen of seminiferous tubules (spermiation) (Figure 5). These four transitions are precisely coordinated in time and space, each occurring in stages VII and VIII of the seminiferous epithelium (Figure 5) [106] (reviewed in [16,111]). The close physical and temporal proximity of each of these transitions, occurring cyclically, with an 8.6-d periodicity in mice [122], suggests a strict coordination. The intimate proximity of each of these transitions is largely conserved in other mammals, including humans [125], rats [112,126], hamsters [127], and rams [127].
在生精上皮的横截面内,生殖细胞的刻板印象聚集或关联发生在分化的不同步骤(图 3图 5)。这些步骤的精确协调称为“生精上皮循环”(或“生精循环”)。在小鼠中,生精周期被细分为12个不同的细胞关联,称为生精(上皮)I至XII期[106]。在精子发生过程中,四个转变指导生殖细胞发育的关键阶段:(i) 精原细胞分化,(ii) 减数分裂起始,(iii) 精子细胞伸长的起始,以及 (iv) 细长的精子细胞释放到生精小管的腔中(精子化)(图 5)。这四个转变在时间和空间上精确协调,每个转变都发生在生精上皮的VII期和VIII期(图5)[106](16,111)中综述)。这些转变在物理和时间上非常接近,周期性地发生,在小鼠中具有 8.6 d 的周期性 [122],表明存在严格的协调性。这些转变的紧密接近性在其他哺乳动物中基本保持不变,包括人类[125]、大鼠[112,126]、仓鼠[127]和公羊[127]。

3.2. Regulation of Spermatogenesis by Vitamin A and RA
3.2. 维生素 A 和 RA 对精子发生的调节

A central role for RA in mammalian spermatogenesis was first described in 1925, when rodents fed a vitamin A-deficient (VAD) diet were found to be sterile [128,129,130] (reviewed in [131,132]). In VAD mice and rats, most germ cells arrest as undifferentiated spermatogonia [133,134,135,136,137]. In VAD rat testes, some germ cells arrest just prior to meiosis, as preleptotene spermatocytes [17,136,138]. When VAD animals are given an injection of all-trans RA, or vitamin A, the arrested spermatogonia undertake differentiation [17,135,137], and the arrested preleptotene spermatocytes initiate meiosis [17]. Further, mice treated daily with WIN18,446—which inhibits the retinaldehyde dehydrogenases (ALDH1A1-3) and thereby prevents local RA production [139,140]—exhibited blocks in both spermatogonial differentiation and meiotic initiation [124,141,142]. Thus, in males, both these premeiotic transitions—spermatogonial differentiation and meiotic initiation—require RA.
RA在哺乳动物精子发生中的核心作用最早于1925年被描述,当时发现喂食维生素A缺乏症(VAD)饮食的啮齿动物是不育的[128,129,130](131,132综述)。在 VAD 小鼠和大鼠中,大多数生殖细胞以未分化精原细胞的形式停滞 [133,134,135,136,137]。在VAD大鼠睾丸中,一些生殖细胞在减数分裂之前停滞,成为前肽素精母细胞[17,136,138]。当 VAD 动物注射全反式 RA 或维生素 A 时,停滞的精原细胞进行分化 [17,135,137],而停滞的前肽素精母细胞开始减数分裂 [17]。此外,每天用WIN18,446治疗的小鼠——它抑制视黄醛脱氢酶(ALDH1A1-3),从而阻止局部RA的产生[139,140]——在精原分化和减数分裂起始中都表现出阻滞[124,141,142]。因此,在雄性中,这两个减数分裂前转变——精原分化和减数分裂起始——都需要 RA。

3.3. The Role of RA and Stra8 at Spermatogonial Differentiation and Meiotic Initiation
3.3. RA 和 Stra8 在精原分化和减数分裂起始中的作用

Stra8, which is required for meiotic initiation, also promotes (but is not strictly required for) spermatogonial differentiation [124]. In the postnatal mouse testis, the STRA8 protein is detected in spermatogonia as early as postnatal day 2 (P2) [47,143], when the first evidence of spermatogonial differentiation occurs [144]. In the adult testis, STRA8 is expressed at spermatogonial differentiation of Aal spermatogonia, and in preleptotene spermatocytes at meiotic initiation; both occur during stages VII–VIII (Figure 4 and Figure 5) [124,145,146]. In mice lacking Stra8, undifferentiated spermatogonia accumulate in unusually high numbers as early as P10 [124], and the remaining germ cells arrest just prior to meiosis, as preleptotene spermatocytes [18,36]. Thus, RA acts instructively, and at least in part through STRA8, at spermatogonial differentiation, distinct from its critical function in meiotic initiation [124].
Stra8 是减数分裂起始所必需的,也促进精原分化(但并非严格要求)[124]。在出生后小鼠睾丸中,早在出生后第 2 天 (P2) [47,143] 就在精原细胞中检测到 STRA8 蛋白,此时精原细胞分化的第一个证据出现 [144]。在成人睾丸中,STRA8 在 Aal 精原细胞的精原分化中表达,在减数分裂开始时在 preleptotene 精母细胞中表达;均发生在VII-VIII期(图4图5)[124,145,146]。在缺乏Stra8的小鼠中,未分化的精原细胞早在P10就以异常高的数量积累[124],而剩余的生殖细胞在减数分裂之前停止,成为前瘦素精母细胞[18,36]。因此,RA 在精原分化中起着指导性作用,并且至少部分通过 STRA8 发挥作用,这与它在减数分裂起始中的关键功能不同 [124]。
Unlike RA deficiency, genetic ablation of Stra8 does not preclude spermatogonial differentiation [124], indicating that RA has additional effects, aside from inducing Stra8 expression, at this transition. Culture experiments [48,147] indicate that treatment of undifferentiated spermatogonia with RA stimulates the expression of Stra8 and of Kit, a marker of spermatogonial differentiation [148,149,150]. In vivo, Kit expression is low in undifferentiated spermatogonia due, in part, to the action of PLZF (also known as ZBTB16). In germ cells, PLZF maintains spermatogonia in an undifferentiated state [151,152] by binding the Kit promoter and repressing its expression [153] (Figure 4). At spermatogonial differentiation, RA induces the expression of its target gene Sall4, which sequesters PLZF from the Kit promoter, thereby increasing Kit expression (Figure 4) [154,155] (reviewed in [156]). RA has also been found to activate the PI3K-AKT-mTOR signaling cascade in a non-genomic manner, stimulating the translation of Kit mRNA [157] (reviewed in [158]). Thus, RA may induce spermatogonial differentiation via several independent pathways, including Stra8, Sall4, and Kit.
与 RA 缺陷不同,Stra8 的基因消融并不排除精原分化 [124],这表明 RA 除了诱导 Stra8 表达外,在这个转变中还有其他作用。培养实验[48,147]表明,RA治疗未分化的精原细胞会刺激Stra8Kit的表达,Kit是精原细胞分化的标志物[148,149,150]。在体内,Kit 在未分化精原细胞中的表达较低,部分原因是 PLZF (也称为 ZBTB16) 的作用。在生殖细胞中,PLZF 通过结合 Kit 启动子并抑制其表达 [153] 将精原细胞维持在未分化状态 [151,152](图 4)。在精原分化时,RA 诱导其靶基因 Sall4 的表达,该基因将 PLZF 与 Kit 启动子隔离开来,从而增加 Kit 的表达(图 4)[154,155](在 [156] 中已论述)。还发现 RA 以非基因组方式激活 PI3K-AKT-mTOR 信号级联反应,刺激 Kit mRNA 的翻译 [157](在 [158] 中已论述)。因此,RA 可能通过几个独立的途径诱导精原分化,包括 Stra8Sall4Kit
During spermatogonial differentiation, RA acts directly on germ cells through RARs. Undifferentiated spermatogonia express several RARs (Figure 4) [159,160], and simultaneous ablation of both RARγ and RARα in germ cells impairs spermatogonial differentiation [159] (reviewed in [145]). Additional targets of RA could be activated indirectly, by the action of RA on the Sertoli cells, as RA signaling via RARα in Sertoli cells is critical for the first round of spermatogonial differentiation [63], and for the differentiation of Sertoli cells at puberty [161].
在精原分化过程中,RA 通过 RAR 直接作用于生殖细胞。未分化的精原细胞表达几种RAR(图4)[159,160],同时消融生殖细胞中的RARγ和RARα会损害精原分化[159]([145]综述)。RA 的其他靶标可以通过 RA 对支持细胞的作用间接激活,因为在支持细胞中通过 RARα 的 RA 信号转导对于第一轮精原细胞分化 [63] 和青春期支持细胞的分化至关重要 [161]。

3.4. Role of RA at the Initiation of Spermatid Elongation and Spermiation
3.4. RA 在精子细胞伸长和精子形成开始时的作用

In the 1980s, Huang and Marshall [162] suggested that vitamin A deficiency may delay spermiation. Moreover, ablation of RARs or RA-synthesizing enzymes (in germ cells and/or Sertoli cells) causes a variety of defects in both meiotic and postmeiotic transitions, including spermiation [63,163,164,165,166,167,168]. A recent study has shown that RA plays primary roles at two postmeiotic transitions; the initiation of spermatid elongation and spermiation (Figure 5) [19]. After injection of the inhibitor WIN18,446, both spermatid elongation and spermiation were delayed, and conversely, a single injection of RA was sufficient to precociously induce both these transitions.
在 1980 年代,Huang 和 Marshall [162] 提出维生素 A 缺乏可能会延迟精精形成。此外,RARs 或 RA 合成酶(在生殖细胞和/或支持细胞中)的消融会导致减数分裂和减数分裂后转换中的各种缺陷,包括精子形成 [63,163,164,165,166,167,168]。最近的一项研究表明,RA 在减数分裂后的两个转变中起主要作用;精子细胞伸长和精子化的开始(图 5)[19]。注射抑制剂 WIN18,446 后,精子细胞伸长和精子化均延迟,相反,单次注射 RA 足以早熟诱导这两种转变。
It remains to be determined whether the requirement for RA at these two post-meiotic transitions is due to the direct action of RA on germ cells, or occurs indirectly, via Sertoli cells. RARs and RXRs are expressed specifically in round spermatids in stages VII and VIII [60], suggesting that RA may act directly on round spermatids to initiate elongation. Indirect RA signaling, via RARs/RXRs in Sertoli cells [60], may also contribute to this process. RA is likely to regulate the release of elongated spermatids indirectly, via Sertoli cells, as these spermatids are thought to be transcriptionally silent (reviewed in [169]). By identifying RA functions in post-meiotic cells, future studies may resolve the mechanism by which RA regulates each of these two postmeiotic transitions.
在这两个减数分裂后转换中对 RA 的需求是由于 RA 对生殖细胞的直接作用,还是间接地通过支持细胞发生,仍有待确定。RAR和RXR在VII期和VIII期的圆形精子细胞中特异性表达[60],表明RA可能直接作用于圆形精子细胞以启动伸长。通过支持细胞中的 RAR/RXR 间接 RA 信号传导 [60] 也可能有助于这一过程。RA 可能通过支持细胞间接调节细长精子细胞的释放,因为这些精子细胞被认为在转录上是沉默的(在 [169] 中综述)。通过识别减数分裂后细胞中的 RA 功能,未来的研究可能会解决 RA 调节这两个减数分裂后转变的机制。

3.5. Source of RA in the Postnatal and Adult Testis
3.5. 产后和成人睾丸 RA 的来源

In postnatal and adult testes, RA-degrading enzymes (Cyp26a1, Cyp26b1, and Cyp26c1) are expressed by peritubular myoid cells that surround the seminiferous tubules [60]. These peritubular myoid cells form a catabolic barrier that prevents RA generated outside of the seminiferous epithelium from reaching the enclosed germ cells [156]. In the seminiferous tubule, RA is produced by two different cellular sources, Sertoli cells and germ cells. Sertoli cells express an RA-synthesizing enzyme, Aldh1a1 [60,170]. Another RA synthesizing enzyme, Aldh1a2, is expressed in pachytene and diplotene spermatocytes from stages VII through XII [60,170]. Indeed, direct quantitation of RA levels confirms that both Aldh1a1-expressing Sertoli cells and the Aldh1a2-expressing germ cells contribute to the total production of RA from circulating retinol [19,171].
在出生后和成人睾丸中,RA降解酶(Cyp26a1Cyp26b1Cyp26c1)由围绕生精小管的肾小管周围肌样细胞表达[60]。这些肾小管周围肌样细胞形成分解代谢屏障,阻止生精上皮外产生的 RA 到达封闭的生殖细胞 [156]。在生精小管中,RA 由两种不同的细胞来源产生,即支持细胞和生殖细胞。支持细胞表达一种RA合成酶Aldh1a1[60,170]。另一种RA合成酶Aldh1a2在VII期至XII期的厚皮细胞和二柱细胞中表达[60,170]。事实上,RA 水平的直接定量证实,表达 Aldh1a1 的支持细胞和表达 Aldh1a2 的生殖细胞都有助于循环视黄醇产生 RA [19,171]。
The RA produced by Sertoli cells is required for spermatogonial differentiation. Sertoli cell-specific ablation of Aldh1a1-3 causes a complete arrest at the first round of spermatogonial differentiation in postnatal mice [63]. In the unperturbed testis, RA from Sertoli cells contributes functionally to both spermatogonial differentiation and meiotic initiation [19]. Recent studies have addressed the question of whether RA produced by pachytene spermatocytes is required for spermatogenesis [19,171,172]. Chemical or genetic depletion of pachytene spermatocytes in adult testes results in delays to both the elongation of the round spermatids and spermiation, but not to spermatogonial differentiation or meiotic initiation [19]. Germ cell-specific ablation of Aldh1a1-3 delays the first round of postnatal spermatogenesis, but these animals show complete spermatogenesis in adult testes at 8 to 10 weeks [171]. The simplest interpretation of these findings is that, in the unperturbed testis, pachytene spermatocytes work collaboratively with Sertoli cells to produce RA levels for the four transitions.
支持细胞产生的 RA 是精原细胞分化所必需的。Aldh1a1-3 的支持细胞特异性消融导致出生后小鼠第一轮精原细胞分化完全停滞 [63]。在未受干扰的睾丸中,来自支持细胞的 RA 在功能上有助于精原细胞分化和减数分裂起始 [19]。最近的研究解决了厚壁精母细胞产生的RA是否是精子发生所必需的问题[19,171,172]。成人睾丸中厚壁精母细胞的化学或遗传耗竭导致圆形精母细胞的伸长和精子形成延迟,但不会延迟精原分化或减数分裂起始[19]。Aldh1a1-3 的生殖细胞特异性消融延迟了出生后第一轮精子发生,但这些动物在 8-10 周时在成人睾丸中表现出完全的精子发生 [171]。对这些发现的最简单解释是,在未受干扰的睾丸中,厚壁精母细胞与支持细胞协同工作,为四个转变产生 RA 水平。
In mice with a Sertoli cell-specific deletion of Aldh1a1-3, the arrest at the first round of spermatogonial differentiation can be rescued by RA injection, as all germ cell cohorts are subsequently observed [63]. Conversely, after injection of RA at 4 weeks of age, Sertoli cell-specific Aldh1a1-3-deficient adults displayed abnormalities in spermiation at 24 weeks of age [63], suggesting that RA from Sertoli cells contributes modestly to this process. Moreover, the level of RA required for spermatogonial differentiation is higher than that required for meiotic initiation [171,172], indicating that each transition is sensitive to the local level of RA. Because the postmeiotic transitions are most sensitive following depletion of RA [19], the postmeiotic transitions may require a higher concentration of RA, from both Sertoli cells and pachytene spermatocytes, than the premeiotic transitions.
在支持 Aldh1a1-3 支持细胞特异性缺失的小鼠中,RA 注射可以挽救第一轮精原细胞分化的停滞,因为随后观察到所有生殖细胞队列 [63]。相反,在 4 周龄注射 RA 后,支持细胞特异性 Aldh1a1-3 缺陷的成人在 24 周龄时表现出精子异常 [63],表明来自支持细胞的 RA 对这一过程的贡献适度。此外,精原分化所需的 RA 水平高于减数分裂起始所需的水平 [171,172],表明每个转变都对 RA 的局部水平敏感。由于减数分裂后转换在 RA 耗竭后最敏感 [19],因此减数分裂后转换可能需要比减数分裂前转换更高浓度的 RA,来自支持细胞和厚纤精母细胞。
When mice lacking Aldh1a1-3 in both Sertoli cells and germ cells are given a single RA injection at P3, some germ cells immediately undergo spermatogonial differentiation and later initiate meiosis (with STRA8 expression) seven days after the injection [171]. Based on this observation, Teletin et al. [171] hypothesized that RA is dispensable for meiotic initiation. However, after a single injection of exogenous RA to postnatal mice, increased levels of RA in the testis are maintained for more than seven days, even under the daily treatment with WIN18,446, which inhibits endogenous RA production [172]. Given that meiotic initiation can be induced by a low threshold of RA [171,172], the injected RA remaining in the seminiferous tubule may be sufficient to induce meiotic initiation in postnatal mice.
当支持细胞和生殖细胞中都缺乏 Aldh1a1-3 的小鼠在 P3 处注射 RA 时,一些生殖细胞立即发生精原分化,并在注射后 7 天开始减数分裂(表达 STRA8)[171]。基于这一观察,Teletin等[171]假设RA对于减数分裂起始是可有可无的。然而,在对出生后小鼠单次注射外源性 RA 后,睾丸中 RA 水平升高维持了 7 天以上,即使在每天使用 WIN18,446 治疗下,WIN18,446 抑制了内源性 RA 的产生 [172]。鉴于 RA 的低阈值可以诱导减数分裂起始 [171,172],残留在生精小管中的注射 RA 可能足以诱导出生后小鼠减数分裂起始。

3.6. Periodicity of Spermatogenesis and RA Levels
3.6. 精子发生和 RA 水平的周期性

In the unperturbed testis, STRA8 is periodically expressed in spermatogonia and is present during the majority of the seminiferous cycle. Specifically, STRA8 is rarely expressed in stages II–VI (before the four transitions), then increases rapidly in stages VII–VIII (during transitions), and remains high thereafter in stages IX–I (Figure 5) [124,145,146]. The expression of STRA8 reflects the presence of RA; when RA levels are increased by injecting RA, or decreased by injecting WIN18,446, STRA8 expression is immediately induced or absent, respectively, in all seminiferous stages, as judged by immunostaining [124]. In good agreement with STRA8 expression, RA concentrations change periodically in the seminiferous tubule [124,146]; absolute quantification of RA levels has found that RA levels are low in stages II–VI, rise in stages VII–VIII, and remain high until stages XII/I (Figure 5) [124]. The expression of RA-metabolizing enzymes may help to explain how this periodicity of RA concentration is established in the adult testis. Aldh1a1 transcripts are present in stages I–VIII in Sertoli cells [156,170], and Aldh1a2 transcripts peak in late pachytene and diplotene spermatocytes in stages VII–XII [60,170]. Thus, Sertoli cell production of RA may precede the germ cell production of RA in each cycle of the seminiferous epithelium. In contrast, the expression of RA storage enzymes, Lrat and Adfp, which function to reduce local RA levels, are detected in stages I–VI/VII [60,170]. Thus, RA concentration in stages II-VI might be kept low, even in the presence of Aldh1a1. Moreover, the CYP26 family of enzymes, which are expressed in Sertoli cells [60,170,173,174], may catabolize RA to maintain tight control of the seminiferous milieu.
在未受干扰的睾丸中,STRA8 在精原细胞中周期性表达,并且存在于生精周期的大部分时间。具体来说,STRA8很少在II-VI期(4个转换之前)表达,然后在VII-VIII期(转换期间)迅速增加,此后在IX-I期保持较高水平(图5)[124,145,146]。STRA8 的表达反映了 RA 的存在;当注射 RA 水平增加或注射 WIN18,446 降低 RA 水平时,根据免疫染色判断,在所有生精阶段分别立即诱导或不存在 STRA8 表达 [124]。与 STRA8 表达一致,RA 浓度在生精管中周期性变化 [124,146];RA水平的绝对定量发现,RA水平在II-VI期较低,在VII-VIII期升高,并且在XII/I期之前一直保持较高水平(图5)[124]。RA 代谢酶的表达可能有助于解释 RA 浓度的这种周期性是如何在成人睾丸中建立的。Aldh1a1 转录本存在于支持细胞的 I-VIII 期 [156,170],Aldh1a2 转录本在晚期厚细胞和双精母细胞的 VII-XII 期达到峰值 [60,170]。因此,在生精上皮的每个周期中,RA 的支持细胞产生可能先于 RA 的生殖细胞产生。相比之下,在I-VI/VII.阶段检测到RA贮存酶LratAdfp的表达,这些酶的作用是降低局部RA水平[60,170]。因此,即使在 Aldh1a1 存在的情况下,II-VI 期的 RA 浓度也可能保持在较低水平。 此外,在支持细胞中表达的 CYP26 酶家族 [60,170,173,174] 可能会分解代谢 RA 以保持对生精环境的严格控制。

3.7. Competence of Germ Cells for Spermatogonial Differentiation
3.7. 生殖细胞的精原分化能力

Despite the persistently elevated RA levels in stages IX–I, spermatogonial differentiation is not observed in these stages (Figure 5). Early undifferentiated As and Apr spermatogonia (found at all stages) and undifferentiated Aal spermatogonia in stages IX–I are unable to express STRA8 in response to RA injection, and do not undergo differentiation (Figure 5) [124]. In the presence of RA, these undifferentiated spermatogonia instead undertake self-renewal and proliferation, which prevents the pool of undifferentiated spermatogonia from being irreversibly depleted. This competence or incompetence for spermatogonial differentiation cannot simply be explained by the expression of RARs, as these receptors are broadly expressed across the seminiferous cycle [60,159,160]. Instead, competence for spermatogonial differentiation is more closely correlated with the proliferative activity of the cells. Specifically, undifferentiated spermatogonia in stages II–VIII, which are competent for differentiation [124], are arrested in the G0/G1 phase of the cell cycle, whereas undifferentiated spermatogonia in stages IX–I are actively proliferating (Figure 5) [107,117]. Further studies are needed to identify the mechanisms that confer competence for spermatogonia to undergo differentiation.
尽管 IX-I 阶段 RA 水平持续升高,但在这些阶段未观察到精原分化(图 5)。早期未分化的 As 和 Apr 精原细胞(在所有阶段发现)和第 IX-I 期的未分化 Aal 精原细胞无法响应 RA 注射而表达 STRA8,并且不会发生分化(图 5)[124]。在 RA 存在的情况下,这些未分化的精原细胞反而进行自我更新和增殖,从而防止未分化精原细胞池不可逆地耗尽。这种精原细胞分化的能力或无能不能简单地用RAR的表达来解释,因为这些受体在整个生精周期中广泛表达[60,159,160]。相反,精原细胞分化的能力与细胞的增殖活性更密切相关。具体来说,具有分化能力的II-VIII期的未分化精原细胞[124]在细胞周期的G0/G1期被停滞,而IX-I期的未分化精原细胞正在积极增殖(图5)[107,117]。需要进一步的研究来确定赋予精原细胞分化能力的机制。

4. Summary and Perspectives
4. 总结和观点

Several lines of evidence support a critical role for RA in directing meiotic initiation in the fetal ovary, and for critical transitions of adult spermatogenesis, including meiotic initiation. In development, embryonic germ cells acquire the competence to initiate meiosis in response to RA. Male germ cells, which escape from RA-induced meiotic initiation in the fetal testis by the catabolism of RA, develop first as undifferentiated spermatogonia, which later acquire competence for spermatogonial differentiation. Male germ cells subsequently acquire competence for meiotic initiation (and possibly initiation of spermatid elongation). These distinct competencies to respond to RA must be strictly regulated. After RA injection, undifferentiated spermatogonia are not competent to initiate meiosis directly; instead, the undifferentiated spermatogonia begin a program of spermatogonial differentiation, followed by six mitotic cell divisions [124]. Further studies of these distinct competencies will help our understanding of the basic mechanisms that govern germ cell development and advance assisted reproduction technologies, such as in vitro gamete production [175,176,177,178].
几条证据线支持 RA 在指导胎儿卵巢减数分裂起始和成体精子发生的关键转变(包括减数分裂起始)中起关键作用。在发育过程中,胚胎生殖细胞获得响应 RA 启动减数分裂的能力。雄性生殖细胞通过 RA 的分解代谢从 RA 诱导的胎儿睾丸减数分裂起始中逃脱,首先发育为未分化的精原细胞,随后获得精原分化的能力。雄性生殖细胞随后获得减数分裂起始(可能开始精子细胞伸长)的能力。必须严格监管这些应对 RA 的不同能力。RA 注射后,未分化的精原细胞不能直接启动减数分裂;相反,未分化的精原细胞开始精原分化程序,然后是 6 次有丝分裂细胞分裂 [124]。对这些不同能力的进一步研究将有助于我们理解控制生殖细胞发育的基本机制,并推进辅助生殖技术,如体外配子的产生[175,176,177,178]。

Author Contributions 作者贡献

Conceptualization, T.E.; writing-original draft and prepared the figures, T.E.; reviewing and editing the manuscript, T.E., M.M.M., P.K.N., D.C.P., and D.G.d.R.
概念化,TE;撰写原始草稿并准备数字,T.E.;审阅和编辑手稿、T.E.、M.M.M.、P.K.N.、D.C.P. 和 D.G.D.R.

Funding 资金

T.E. is supported by Japan Society for the Promotion of Science (JSPS) KAKENHI, Grant Number JP19K06439. M.M.M. is supported by a NRSA Postdoctoral Fellowship from the NICHD, Grant Number F32HD093391. D.C.P is an Investigator of the Howard Hughes Medical Institute.
TE 得到了日本科学振兴会 (JSPS) KAKENHI 的支持,资助号 JP19K06439。MMM 得到了 NICHD 的 NRSA 博士后奖学金的支持,资助号 F32HD093391。D.C.P 是霍华德休斯医学研究所的研究员。

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Anterior-to-posterior wave of Dazl and Stra8 expression from E10.5 to E14.5 in mouse fetal gonads. Germ cells are shown in circles, with cells expressing Dazl shown in orange, and cells expressing Stra8 and Dazl shown in blue. After gonadal colonization, germ cells continue to proliferate until E13.5 [22]. In the fetal mouse testis, germ cells become enclosed by somatic cells, with testis cords formed between E12.5 to E14.0 [14,23].
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Figure 2. Diagram of germ cell development in mouse fetal gonads of both sexes. Red box: female gonad (ovary). Blue box: male gonad (testis). DAZL, STRA8, REC8, and NANOS2 are expressed in germ cells. ALDH1A1 and CYP26B1 are expressed in fetal gonads. ALDH1A2 and ALDH1A3 are expressed outside the gonads.
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Figure 3. Structure of the mouse testis comprising seminiferous tubules. In any given tubule cross-section, one observes germ cells at different steps of their development into elongated spermatids. These germ cell types are concentrically layered; undifferentiated spermatogonia lie on the basal lamina of the tubule, and germ cells move toward the tubule lumen as they differentiate [106]. Germ cell differentiation is precisely timed; hence, particular steps of development are always found together in close physical proximity. Blue line indicates the orientation of testis cross-sections. A representative tubule cross-section in stage VII–VIII, stained with hematoxylin and periodic acid-Schiff (He-PAS), is shown with grayscale version. Star: Sertoli cell nucleus. White arrowhead: type spermatogonium. Dots: preleptotene (red) spermatocytes, pachytene spermatocytes (yellow), and step 7–8 round spermatids (green). Brown area: elongated spermatids. Scale bar = 30 μm.
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Figure 4. Multiplication of undifferentiated spermatogonia and spermatogonial differentiation. Upon division, the Asingle (As) spermatogonia can self-renew and produce two new singles or the daughter cells, Apaired (Apr) spermatogonia, remain connected by an intercellular bridge. The Apr spermatogonia subsequently divide further into chains of 4, 8, or 16 cells, called Aaligned (Aal) spermatogonia that undergo spermatogonial differentiation (purple) in response to RA. As, Apr, and Aal spermatogonia are referred to as undifferentiated spermatogonia. After the spermatogonial differentiation, Aal spermatogonia transit into A1 differentiating spermatogonia without a mitotic division [114]. Expression patterns of PLZF, RARγ, STRA8, SALL4, and KIT are indicated as solid lines.
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Figure 5. Diagram of mouse spermatogenesis. Oakberg [106] identified 12 distinct cellular associations, called seminiferous stages I–XII. It takes 8.6 days for a section of seminiferous tubule, and the germ cells contained within, to cycle through all 12 stages [122]. Four turns of this seminiferous cycle are required for a germ cell to progress from undifferentiated spermatogonium to spermatozoon. As, Apr, and Aal: Asingle, Apaired, and Aaligned spermatogonia. A1–A4: A1–A4 differentiating spermatogonia. In, and B: intermediate and type B spermatogonia. Pl, L, Z, P, D, and SC2: preleptotene, leptotene, zygotene, pachytene, diplotene, and secondary spermatocytes. Steps 1–16: steps in spermatid differentiation. Purple: germ cells undergoing spermatogonial differentiation; green: meiotic initiation; brown: initiation of spermatid elongation; gray: release of elongated spermatids. Black box: population of undifferentiated spermatogonia. Gray box: the leptotene spermatocytes undergoing migration of basal to luminal compartment [123]. Dark blue: stage with high RA concentration. Light blue line: STRA8 expression in the unperturbed testis. Dashed light blue line: STRA8 expression induced by RA injection in undifferentiated spermatogonia. (After RA injection, undifferentiated Aal spermatogonia in stages II–VI precociously express STRA8 [124]).
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Endo, T.; Mikedis, M.M.; Nicholls, P.K.; Page, D.C.; de Rooij, D.G. Retinoic Acid and Germ Cell Development in the Ovary and Testis. Biomolecules 2019, 9, 775. https://doi.org/10.3390/biom9120775

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Endo T, Mikedis MM, Nicholls PK, Page DC, de Rooij DG. Retinoic Acid and Germ Cell Development in the Ovary and Testis. Biomolecules. 2019; 9(12):775. https://doi.org/10.3390/biom9120775

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Endo, Tsutomu, Maria M. Mikedis, Peter K. Nicholls, David C. Page, and Dirk G. de Rooij. 2019. "Retinoic Acid and Germ Cell Development in the Ovary and Testis" Biomolecules 9, no. 12: 775. https://doi.org/10.3390/biom9120775

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