Molecular genetics of sex determination

In humans, the choice between male or female development is genetically determined. Sex determination take place when the bipotential embryonic gonad becomes either testis or ovary. This process is directed by genes that have been discovered by genetic analysis of sex-reversed patients and confirmed by knockout experiments in mice. The testis-determining pathway is better known than the ovary pathway. SRY, a gene located on the Y chromosome, triggers a complex genetic cascade leading to testicular differentiation. In this cascade, two genes play a crucial role in male differentiation, SOX9 and FGF9, which contribute to testicular cord formation. However, only a minority of 46,XY sex-reversed patients can be explained by mutations in known genes such as SRY, SOX9, WTI, and SF1, suggesting that other genes influencing sex determination are yet to be discovered. In females, some rare genes that induce ovarian failure or female-to-male sex reversal have been found through gene-targeted inactivation in mice or positional cloning of mutations in humans and goats. In both sexes, genetic analysis of sex-reversed individuals (XX males, XX and XY hermaphrodites, and XY with complete or partial dysgenesis) remains an approach of choice to isolate new genes involved in sex determination.     

The Y chromosome and its role in testis differentiation and spermatogenesis

The Y chromosome contains genes and gene families that play critical roles in the processes of testis determination and testis differentiation. Great strides have been made toward defining the genetic pathways associated with the determination of gender. The data are summarized and discussed and clinical ramifications are considered.     

哺乳动物P1精蛋白基因表达的研究

目的:研究哺乳动物P1精蛋白基因在大鼠和小鼠中的表达。方法:用RT-PCR法制备大鼠P1精蛋白(rP)cDNA,用小鼠P1精蛋白(mP1)DNA重组质粒(pUC8)制备mP1cDNA,rPcDNA和mP1cDNA经迪高辛标记后作为探针,用Northern杂交等技术分析基因表达情况。结果:rPcDNA与大鼠睾丸RNA有杂交反应,而与肝、脑RNA无交叉反应;rPcDNA和小鼠睾丸RNA有交叉反应;mP1cDNA探针与性成熟不同阶段小鼠睾丸RNA杂交结果表明:小鼠性成熟过程中,在睾丸出现圆形精子细胞后mP1基因开始转录,而翻译成蛋白质是在性成熟小鼠睾丸内出现长形精子细胞后。结论:哺乳动物P1精蛋白的表达有器官特异性,是睾丸特异表达的基因,P1精蛋白基因在进化上保守,因而在大、小鼠睾丸中均能检测到杂交讯号;精蛋白基因的表达表现为翻译延迟,该基因在圆形精子细胞阶段开始转录,而在长形精子细胞阶段翻译成蛋白质。     

Epigenetics of sex determination in mammals

Epigenetics is the study of changes in gene function that cannot be explained by changes in DNA sequence. A mammalian body contains more than two hundred types of cells. Since all of them are derived from a single fertilized egg, their genotypes are identical. However, the gene expression patterns are different between the cell types, indicating that each cell type has unique own “epigenotype”. Epigenetic gene regulation mechanisms essentially contribute to various processes of mammalian development. The essence of epigenetic regulation is the structural change of chromatin to modulate gene activity in a spatiotemporal manner. DNA methylation and histone modifications are the major epigenetic mechanisms. Sex determination is the process for gender establishment. There are two types of sex-determining mechanisms in animals, environmental sex determination (ESD) and genotypic sex determination (GSD). Recent studies have provided some evidence that epigenetic mechanisms play indispensable roles in ESD and GSD. Some fishes undergo ESD, in which DNA methylation is essentially involved. GSD is employed in therian mammals, where Sry (sex-determining region on the Y chromosome) triggers testis differentiation from undifferentiated gonads. Sry expression is tightly regulated in a spatiotemporal manner. A recent study demonstrated that histone modification is involved in Sry regulation. In this review, we discuss the role of epigenetic mechanisms for sex determination in mammals and other vertebrates.     

Characterization of lactoferrin receptor on human spermatozoa

Lactoferrin (LF) is abundant in human seminal plasma and on sperm surfaces. However, lactoferrin receptor (LFR) on human spermatozoa has not yet been reported. To study the expression, localization and characteristics of LFR on human spermatozoa, different experimental approaches were applied: LFR gene was amplified from a human testis cDNA library and recombinant LFR (rLFR) protein was produced in the expression vector Escherichia coli BL21 (DE3); human sperm membrane proteins were extracted and analysed via Western blot; the binding of LF to LFR was investigated by Far-Western blot, immunoprecipitation and autoradiography analysis and the localization of LFR on sperm surfaces was detected using immunofluorescence. LFR gene was amplified from a human testis cDNA library and the molecular weight of rLFR was 34kDa. The native LFR on human spermatozoa was a 136-kDa tetramer which was anchored to the sperm head and mid-piece through glycophosphatidylinositol. LF could bind to LFR competitively in vitro. As far as is known, this study has elucidated for the first time that LFR was expressed at the testis level, was anchored to the sperm membrane by glycophosphatidylinositol during spermatogenesis. LFR may play important roles through binding to and mediating LF.     

Impaired testosterone biosynthesis in cryptorchidism

In an attempt to determine whether the production and synthesis of testosterone (T) by the testis is impaired by the cryptorchid state, the ability of the cryptorchid rat testis to form T was assessed at various time periods into adulthood after the surgical induction of cryptorchidism in the newborn period. The intratesticular T content of the descended testis rose from 0.3 ng/testis at 14 days of age to 71.2 ng/testis at adulthood (day 56); whereas in the cryptorchid testis, the values were 0.4 ng/testis and 2.0 ng/testis, respectively, at the same ages (P less than 0.001). For determination of the site of inhibition of T biosynthesis in the cryptorchid testis, the enzymatic activities (17 alpha-hydroxylase, 17,20-desmolase, and 17 beta-hydroxysteroid dehydrogenase) in the delta 4T biosynthetic pathway were measured. All these enzyme activities in the cryptorchid testis were inhibited at 56 days of age by about 80% when compared with the descended testis (P less than 0.01). These data suggest that cryptorchidism exerts a deleterious effect on the ability of the Leydig cells to synthesize T and may in part explain the abnormal morphology and resultant infertility seen in patients with cryptorchidism.     

一例罕见SRY阴性46,XX,inv(9)男性

目的:探讨46,XX男性性反转综合征的临床表现和遗传学基础。方法:对1例46,XX,inv(9)(p11q13)男性不育患者进行系统检查,包括精液分析、血清激素和性腺组织活检,PCR法分析SRY基因和Y染色体上包括AZF区域的23个特异性位点,用FISH技术进一步证实患者核型及SRY的缺失,同时检测与性腺分化相关的SOX9和DMRT1基因内部的STR位点,以观察有无基因剂量的改变。结果:外周血淋巴细胞染色体核型分析为46,XX,inv(9)(p11q13)。Y染色体上SRY和其它23个特异位点均为阴性。未发现SOX9和DMRT1基因剂量改变。性腺组织活检证实为发育不良的睾丸组织。由于睾丸组织DNA模板提取失败,故未能检测睾丸组织的特异性位点。结论:本病例的性反转不是由SRY基因转移引起的。性别决定和分化的机制目前还不完全清楚,除了SRY等已知的基因外,可能还存在其它未知但重要的性别决定基因。     

Sexual differentiation

Sexual differentiation in humans is genetically and hormonally controlled. In response to a signal from a dominant-acting gene on the Y chromosome, primordial cells in the embryonic gonad ridge differentiate into Sertoli cells and affect newly migrated germ cells to differentiate as spermatogonia, thus creating a testis. The cells of the embryonic testis secrete hormones that lead to the development of most, if not all, male secondary sexual characteristics. The Sertoli cells secrete müllerian inhibitory factor (MIF), causing regression of the müllerian ducts and of stray oogonia. The Leydig cells secrete testosterone, causing differentiation and growth of the wolffian duct structures. Dihydrotestosterone, created by metabolism of testosterone, causes growth of the prostate and phallus and fusion of the labioscrotal folds. In the absence of SRY, Sertoli cell differentiation does not occur. Rather germ cells migrating into the primordial gonad differentiate as oogonia and cause interstitial cells to differentiate as granulosa cells. In the absence of MIF and testosterone, the müllerian ducts differentiate and grow as female internal genitalia and the external genitalia are feminized. Several genes have been identified that control testis determination. These include SRY, WT1, SOX9, SF1, XH2, and DAX1. Most of these genes were discovered by analysis of rare cases of sex reversal (genetic sex of one type, gonadal sex of the other type).     

γ-氨基丁酸(GABA_A)样受体基因在大鼠睾丸存在的初步研究

为探讨大鼠睪丸是否存在GABA样受体并了解它在不同发育时期的表达情况,本实验将大鼠睪丸的mRNA微注射进入爪蟾卵母细胞表达;电压钳记录结果显示:用500μmol/L浓度GABA溶液灌流可引发其产生约30nA的内向电流.表明大鼠睪丸可能存在GABA受体,用GABA_A 受体抑制剂Bicuculine和Picrotoxin可抑制GABA的这种作用.用GABA_A 受体激动剂Muscimol溶液灌流可引发内向电流产生,而用GABA_B受体激动剂Baclofen则无此效应,表明睪丸组织存在的GABA受体主要为A亚型而非B亚型.基于上述结果,本实验根据神经系GABA受体亚基多肽链第二跨膜区的.cDNA保守序列合成探针,用与成年大鼠脑和各生长发育时期大鼠睪丸RNA进行斑点(dot)杂交分析,结果显示成年大鼠睪丸比其大脑有较强的杂交信号,表明睪丸的受体RNA可能比脑组织GABA受体RNA与探针序列有较多的同源区段,提示该受体可能是一种类似于GABA的受体,即GABA_A样受体(GABA_A like receptor).同时,斑点杂交结果还进一步显示不同发育时期大鼠睪丸GABA_A样受体表达有差异,基本规律是新生大鼠表达量极少,出生30d至100d时表达量最高,200d后表达开始减少.     

新基因TSBAG的生物信息学分析及其在小鼠睾丸组织中的表达

目的：筛选与精子发生相关的睾丸特异性新基因,并探讨其在精子发生过程中所起的作用。方法：将不同发育阶段的小鼠睾丸组织cDNA探针与Affymetrix全基因组芯片进行杂交,通过杂交信号的比较,筛选出差异表达的基因。用生物信息学软件对该基因进行生物信息学分析。RT-PCR分析该基因在小鼠不同发育阶段睾丸、小鼠不同组织中的表达。结果：通过4日龄、9日龄、18日龄、35日龄、54日龄和6月龄小鼠睾丸的芯片信号比较筛选出一个差异表达功能未知的新基因(GenBank登录号：BC049770),全长891 bp,含有702 bp的完整ORF,编码一个233个氨基酸、相对分子量为26．938 kD的蛋白质,我们将其命名为TSBAG(testis-specific BAG-like protein)。亚细胞定位预测显示,TSBAG基因可能在细胞核中表达。EBI功能域预测发现,在蛋白质序列8-62处有一个BAG功能域。RT-PCR分析表明,TSBAG基因特异性表达于小鼠睾丸组织中且具有时序性表达。TSBAG蛋白在人的同源基因GenBank登录号为AY349359,在233个氨基酸区域内有83%的同源性。结论：TSBAG基因在小鼠睾丸组织中特异性表达,且与小鼠精子发生过程一致。     

Isolation of a phylogenetically conserved and testis-specific gene using a monoclonal antibody against the serological H-Y antigen.

Several cDNA clones of a gene termed male-enhanced antigen-2 (Mea-2), have been isolated from a mouse testicular expression cDNA library using a monoclonal histocompatability Y (H-Ys) antibody which detects specific protein(s) present in the mouse testis but not the ovary. The Mea-2 gene is phylogenetically conserved among various mammalian species examined, and is expressed at high levels in adult mouse testis. The expression pattern of Mea-2 is very similar to that of another gene, the male-enhanced antigen-1 (Mea-1), previously isolated using a polyclonal H-Ys antibody. Northern blotting and RT-PCR analyses demonstrated that Mea-2 is also expressed in other adult and fetal mouse organs at low levels. The testis-enhanced expression of this gene is associated with germ cell development at mid- to late-meiotic stages of spermatogenesis. Analysis of an intersubspecies mouse backcross has assigned this gene to chromosome 5, between the loci Gus and Hnf-1.     

Quantitation of sex chromosomal influence(s) on the somatic growth of fetal gonads in vivo

Matings between mice carrying the heterozygous autosomal gene Steel produce 25% homozygous Sl/Sld mutants having agametic gonads composed of somatic tissue alone. Mutants show a normal 1:1 male:female sex ratio and gonadal sex corresponds to genotypic sex. Gonads from both normal and mutant male and female fetuses were obtained on a daily basis from the twelfth day of gestation to birth and morphometrically evaluated for determination of the quantitative expression of XY and XX chromosomal combinations on growth of gonadal somatic tissue. Growth of testicular soma was found to be independent of a germ cell influence throughout the entire course of gestation whereas a germ cell influence on growth of ovarian gonadal soma was apparent from day 18 and thereafter. By birth, testicular somatic growth displayed a ten-fold increase over that of the ovary. These results suggest that early gonadal somatic growth is independent of a germ cell influence. The significant impact of a Y-chromosomal influence on gonadal somatic growth may suggest that male-specific gene product(s) regulate growth as well as differentiation of the somatic elements of the fetal testis.     

Development and regulations of testicular functions in the human foetus

Two major functions are assumed by the testis: the production of male gametes (that is, spermatozoa) and the production of steroid hormones. Both two functions are established during fetal life and are essential to the adult fertility and the masculinization of the internal tract and genitalia. For many years, our laboratory has been interested in the ontogeny of those two functions in rodents and, since 2003, in collaboration with gynecology and obstetrics service of professor R. Frydman in Antoine-Béclère hospital, we have studied them in human. The first aim of this work was to improve the global knowledge of the human fetal testis development by using both our experimental data and the literature. Then, we focused on the different defects that can occur during the fetal testis development. Indeed, male reproductive abnormalities have been steadily increasing since the last decades and are thought to be related to the concomitant increase of the concentration of contaminants and particularly of endocrine disruptors in the environment. Thus, we decided to study the effect of endocrine disruptors on human fetal testis and, more particularly, the effect of phthalates, by using an organ culture system developed for human. In contrast to the data obtained in rat, mono (ethylhexyl)-phthalate (MEHP), an active metabolite of the most widespread phthalate in the environment, does not disturb the steroidogenic function. On the other hand, it has a negative effect on the male germ cells number. This study is the first experimental demonstration of a negative effect of phthalates directly on human fetal testis.     

Role of INSL3 and LGR8 in cryptorchidism and testicular functions

Cryptorchidism is the most frequent congenital anomaly of the urogenital tract in human males. INSL3 and LGR8/GREAT proteins seem to act as ligand and receptor respectively, and to have a role in gubernaculum development involved in testicular descent. Mutations in the INSL3 gene or LGR8/GREAT were found to be associated with cryptorchidism in humans. In a cohort of 135 ex-cryptorchid patients and 100 controls, mutations were sought in INSL3 and LGR8/GREAT genes by sequencing. Six patients were found with mutations in the INSL3 gene and four patients with LGR8/GREAT mutation (10/135, 7.4%). The 10 patients show different phenotypes, ranging from normozoospermia to complete azoospermia, and from bilateral cryptorchidism to retractile testes. Furthermore, the endocrine function of the testis appeared normal in all subjects. These findings demonstrate that INSL3-LGR8/GREAT mutations are frequently associated with human cryptorchidism, and that the only clinical consequence of alterations of the INSL3-LGR8/GREAT system seems to be failure of the testis to descend normally in the scrotum during embryonic development, without affecting the spermatogenic and endocrine components of the testis itself. The first analysis in humans of INSL3 was then performed using a novel radioimmunoassay kit to measure INSL3 concentrations in serum of adults. The results show that INSL3 circulates in adult men, it is a male-specific hormone, and it is of almost exclusively testicular origin. The role of this hormonal system in adulthood is, however, to date unknown.     

Local regulation of T cell numbers and lymphocyte-inhibiting activity in the interstitial tissue of the adult rat testis

The testis is an immunologically privileged site, and transplantation data suggest that this privilege may be enhanced in cryptorchidism. Although alphabetaT cells, which mediate and promote the immune response, have access to the normal testis, relatively little is known about these cells in the abdominally located testis. An increase in testicular lymphocyte-inhibiting cytokines has also been implicated in enhanced graft survival following the experimental induction of cryptorchidism. Consequently, T cell traffic and lymphocyte-inhibiting activity in testes of cryptorchid adult rats were examined in the following study. Numbers of alphabetaT cells and the cytotoxic CD8(+) T cell subset in the testis were unaffected following 1 month of cryptorchidism. In contrast, subcutaneous testosterone implants, which inhibit Leydig cell function through suppression of gonadotrophin secretion, reduced these parameters in both scrotal and abdominal testes. Testicular T cell numbers were positively correlated with the number of testicular resident macrophages, which also were reduced by subcutaneous testosterone implants. The concentration of lymphocyte-inhibiting activity in the testicular interstitial fluid was reduced by 80% in short-term (1 month) and longer-term (3 months) cryptorchidism. These data indicate that the T cell population, and in particular the CD8(+) T cell subset, in the rat testis is functionally related to the resident macrophages or Leydig cells. On the other hand, testicular lymphocyte-inhibiting activity does not appear to be a determinant of the number of testicular T cells, and may not be a major factor in the prolonged survival of certain grafts in the abdominal testis.