Testicular Somatic Cells Research Articles

Phagocytic removal of apoptotic spermatogenic cells by Sertoli cells: mechanisms and consequences.

More than half of differentiating spermatogenic cells undergo apoptosis before maturing into spermatozoa during mammalian spermatogenesis. These cells are selectively and rapidly eliminated through phagocytosis by Sertoli cells, a testicular somatic cell type possessing phagocytic activity. We have investigated the mechanism by which Sertoli cells specifically recognize and phagocytose apoptotic spermatogenic cells and the consequences of phagocytosis. We showed by in vitro as well as in vivo analyses that Sertoli cells recognize apoptotic spermatogenic cells through the binding of their surface receptor, class B scavenger receptor type I, to phosphatidylserine that is expressed on the surface of spermatogenic cells during apoptosis. The inhibition of phagocytosis in live animals resulted in a decrease in the number of epididymal sperm. These results suggest that phosphatidylserine-mediated phagocytosis of apoptotic spermatogenic cells by Sertoli cells is required for the efficient production of sperm.

The role of GATA in mammalian reproduction.

GATA transcription factors are emerging as critical players in mammalian reproductive development and function. GATA-4 contributes to fetal male gonadal development by regulating genes mediating Müllerian duct regression and the onset of testosterone production. GATA-2 expression appears to be sexually dimorphic being transiently expressed in the germ cell lineage of the fetal ovary but not the fetal testis. In the reproductive system, GATA-1 is exclusively expressed in Sertoli cells at specific seminiferous tubule stages. In addition, GATA-4 and GATA-6 are localized primary to ovarian and testicular somatic cells. The majority of cell transfection studies demonstrate that GATA-1 and GATA-4 can stimulate inhibin subunit gene promoter constructs. Other studies provide strong evidence that GATA-4 and GATA-6 can activate genes mediating gonadal cell steroidogenesis. GATA-2 and GATA-3 are found in pituitary and placental cells and can regulate alpha-glycoprotein subunit gene expression. Gonadal expression and activation of GATAs appear to be regulated in part by gonadotropin signaling via the cyclic AMP-protein kinase A pathway. This review will cover the current knowledge regarding GATA expression and function at all levels of the reproductive axis.

Effects of Adenovirus Mediated Gene Transfer to Mouse Testis In Vivo on Spermatogenesis and Next Generation

We directly injected DNA into mouse testes in vivo using an adenovirus vector to transfect testicular cells. We then analyzed the transfection efficiency, immunological problems and effects of gene transfer on spermatogenesis and the next generation. In this study we discuss the potential of gene therapy for male infertility. A replication incompetent human adenovirus serotype 5 contained 2 deletions (E1 and E3 deletions) and was constructed such that the transgene was driven by the chicken beta-actin promoter to promote over expression of the downstream target gene (Lac Z). This adenovirus vector or control solution was injected into the interstitial space (intratesticular injection) or seminiferous tubules (intratubular injection) of the mouse testis. We investigated beta-galactosidase gene expression by X-gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside) staining, the effects of gene transfer on spermatogenesis by evaluating the frequency of apoptotic cells by the TUNEL method, the inflammatory response on testes by detecting CD4 and CD8 positive cells immunohistochemically, and interleukin (IL)-6 and IL-8 by immunoblot analysis, epididymides sperm motility and the reproductive response of each mouse 3, 7, 14 and 28 days after injection. Intratesticular injection of adenovirus vector resulted in strong transgene expression in Leydig cells. In contrast, intratubular injection resulted in strong expression in Sertoli cells. Transgene expression was not detected in germ cells by either method. The peak of beta-galactosidase activity was on day 7, ie 0.674 +/- 0.20 (intratesticular) and 0.534 +/- 0.22 U (intratubular), and it decreased with time thereafter. The apoptosis index on day 7 was significantly higher in adenovirus injected groups than in noninjected groups, ie 0.46 +/- 0.20 vs 0.10 +/- 0.11 (intratesticular) and 0.78 +/- 0.31 vs 0.24 +/- 0.10 (intratubular). Transfected animals showed a slight mononuclear inflammatory response in the testes composed of CD4 and CD8 positive cells. Adenovirus vector stimulation resulted in the induction of IL-6 and IL-8 secretion in the testis. These immune responses subsided after day 7. There were no significant differences in the percent of motile sperm or the rate of abnormal sperm between the groups on any day after injection. Reproductive ability remained almost normal even after adenovirus mediated gene transfer with no effect observed in offspring. Our results suggest that although slight spermatogenic damage and inflammatory response caused by these methods may present problems, adenovirus mediated gene transfer may be effective for transfecting testicular somatic cells and applicable for in vivo gene therapy for male infertility in the future.

Differential expression of succinyl CoA transferase (SCOT) genes in somatic and germline cells of the mouse testis

Succinyl CoA:3-oxo acid CoA transferase (SCOT/OXCT; EC 2.8.3.5) is a key mitochondrial enzyme in the metabolism of ketone bodies in various organs (but not in the liver). We identified a cDNA clone of the testicular germ cell-specific succinyl CoA transferase isozyme (SCOT-t). We then isolated a mouse orthologue of the SCOT/OXCT cDNA (SCOT-s) and determined the expression of the two types of SCOT in the testis. The mRNAs of scot-s and scot-t were expressed exclusively in testicular somatic cells (i.e. Leydig and Sertoli cells) and germ cells, respectively. SCOT enzymatic activities were assayed in Leydig cell (SCOT-s) and sperm (SCOT-t) fractions. The SCOT activity in sperm was 2.5-fold higher than that in Leydig cells. We conclude that germ cells and somatic cells differentially express the SCOT enzymes and that the SCOT activity of sperm caused exclusively by SCOT-t should play an important role in sperm activity.

Multidrug resistance genes and p-glycoprotein in the testis of the rat, mouse, Guinea pig, and human.

Study of the multidrug resistance phenomenon in tumor cell lines has led to the discovery of the product of the multidrug resistance (MDR) type 1 genes, the plasma membrane P-glycoprotein (P-gp) that functions as an energy-dependent pump for the efflux of diverse anticancer drugs. P-gp was also recently identified in normal epithelial cells with secretory/excretory functions and in the endothelial cells of the capillary blood vessels in the brain and the testis. These endothelial cells are key elements of the blood-brain and blood-testis barriers, respectively. The aim of this study, in the rat, mouse, guinea pig, and human, was to determine whether testicular cells other than the capillary endothelial cells could express MDR type I genes. Immunohistochemistry on testicular sections revealed that P-gp is present in interstitial cells in the mouse, rat, and human testes, in early and late spermatids in guinea pig testis, and in late spermatids in the rat, mouse, and human. Reverse transcription-polymerase chain reaction analysis on isolated mouse, rat, and human cells showed that all somatic testicular cells (Leydig cells, macrophages, peritubular cells, and Sertoli cells) and the cytoplasmic lobes from rat late spermatids expressed MDR type I mRNAs, whereas spermatogonia, pachytene spermatocytes, and early spermatids did not. An ontogenesis study in the mouse reveals that type I MDR gene expression begins at 13.5 days postcoitum at the time when the seminiferous cords and the blood vessels appear and are maintained thereafter. Finally, two functional tests on isolated rat cells, the doxorubicin and rhodamine uptake assays, demonstrated that rat testicular macrophages, Leydig cells, peritubular cells, and Sertoli cells displayed a multidrug-resistance activity, whereas spermatogonia, pachytene spermatocytes, and early spermatids did not. Western blot experiments have revealed that a P-gp of 175 kDa is present in the human testis as well as in the rat Leydig cells, testicular macrophages, peritubular cells, and Sertoli cells, but is absent in spermatogonia, spermatocytes, and early spermatids. We conclude that P-gp is involved in the self-protection of the somatic cells and is most probably one of the molecules that confers its functionality to the blood-testis barrier. The absence of expression of MDR type I genes in mitotic and meiotic germ cells probably explains their particular vulnerability to various anticancer drugs. In contrast, expression of the P-gp in the haploid cells most likely reflects the ability of spermatozoa to assume their own antidrug defense.

Apoptosis and Proliferation of Human Testicular Somatic and Germ Cells during Prepuberty: High Rate of Testicular Growth in Newborns Mediated by Decreased Apoptosis

Programmed cell death and proliferation are evolutionary conserved processes that play a major role during normal development and homeostasis. In the testis, during the fetal and newborn periods, they might determine final adult size and fertility potential. In the present study, we have measured the relative number of testicular cells in apoptosis and in active proliferation in the seminiferous cords and in the interstitium, at different age periods of prepubertal testicular development in humans. Testes from 44 prepubertal subjects without endocrine and metabolic abnormalities were collected at necropsy. They were divided in three age groups (Gr): Gr 1, newborn (1- to 21-d-old neonates), n = 18, mean (+/-SD) age 0.3 +/- 0.23 months; Gr 2, post natal activation (1- to 6-month-old infants), n = 13, mean age 3.93 +/- 1.90 months; and Gr 3, early childhood period (1- to <6-yr-old boys), n = 13, mean age 31.5 +/- 18.9 months. Apoptosis was detected in 5- microm tissue sections using a modified terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and cell proliferation was assessed by Ki-67 immunohistochemistry. Evaluation of apoptosis was confirmed by estimation of active caspase-3. Mean (+/-SD) testicular weight was 0.38 +/- 0.20, 0.54 +/- 0.35, and 0.51 +/- 0.11 g in Gr 1, Gr 2, and Gr 3, respectively. In Gr 1, there was a significant positive correlation between age and testis weight (P = 0.02). Mean (+/-SD) germ cell apoptotic index, AI, (% of apoptotic cells out of total cell number) was 15.0 +/- 6.60, 27.0 +/- 8.80 and 33.4 +/- 11.4 in Gr 1, Gr 2, and Gr 3, respectively. In Sertoli cells, it was 6.60 +/- 4.07, 22.0 +/- 14.0 and 27.5 +/- 19.8, respectively. In interstitial cells, it was 10.2 +/- 6.38, 18.0 +/- 6.70 and 25.7 +/- 15.5, respectively. In the three types of cells, AI in Gr 1 was significantly lower than in Gr 2 or Gr 3 (P < 0.05). Mean (+/-SD) germ cell proliferation index, PI, was 18.6 +/- 13.0, 10.0 +/- 6.50 and 10.9 +/- 6.24% in Gr 1, Gr 2, and Gr 3, respectively. In Sertoli cells and in interstitial cells PI was similar in the three age groups. The PI/AI ratio was used to compare relative differences among age groups. The PI/AI ratio of germ cells, Sertoli cells and interstitial cells in Gr 1 was significantly higher than in Gr 2 or Gr 3 (P < 0.05). It is concluded that, in normal subjects, there is a vigorous growth of the testis during the newborn period with subsequent stabilization during the first years of prepuberty. This cell growth seems to be mainly mediated by decreased apoptosis. The factors that modulate apoptosis of testicular cells are not known, but it is remarkable that this change takes place before the testosterone peak of the post natal gonadal activation of the first trimester of life. These changes taking place during the newborn period might be important to define testicular function in adults.

Sex reversal of genetic females (XX) induced by the transplantation of XY somatic cells in the medaka, Oryzias latipes.

In order to investigate the function of gonadal somatic cells in the sex differentiation of germ cells, we produced chimera fish containing both male (XY) and female (XX) cells by means of cell transplantation between blastula embryos in the medaka, Oryzias latipes. Sexually mature chimera fish were obtained from all combinations of recipient and donor genotypes. Most chimeras developed according to the genetic sex of the recipients, whose cells are thought to be dominant in the gonads of chimeras. However, among XX/XY (recipient/donor) chimeras, we obtained three males that differentiated into the donor's sex. Genotyping of their progeny and of strain-specific DNA fragments in their testes showed that, although two of them produced progeny from only XX spermatogenic cells, their testes all contained XY cells. That is, in the two XX/XY chimeras, germ cells consisted of XX cells but testicular somatic cells contained both XX and XY cells, suggesting that the XY somatic cells induced sex reversal of the XX germ cells and the XX somatic cells. The histological examination of developing gonads of XX/XY chimera fry showed that XY donor cells affect the early sex differentiation of germ cells. These results suggest that XY somatic cells start to differentiate into male cells depending on their sex chromosome composition, and that, in the environment produced by XY somatic cells in the medaka, germ cells differentiate into male cells regardless of their sex chromosome composition.

Transmeiotic differentiation of zebrafish germ cells into functional sperm in culture.

Because cell culture systems are easily accessible for experimental genetic manipulation, male germ cell culture is of great usefulness in creating sperm vectors. This report describes that cultured male germ cells of zebrafish (Danio rerio) underwent mitosis and transmeiotic differentiation, including the entire process of meiosis, to develop into functional sperm. Enzymatically dissociated testicular cells containing germ cells were co-cultured on feeder cells derived from tumor-like testis, which exhibited features characteristic of Sertoli cells such as phagocytic activity and transcription of the Wilms' tumor suppressor wt1 and sox9a genes. Germ cells formed a clump, divided by mitosis, and differentiated into flagellated sperm on the feeders. Expression of the germ cell marker gene vas was prolonged in co-culture with the feeders, compared with culture of dissociated testicular cells alone, indicating that the feeder cells stimulate proliferation of spermatogonia. When cultured germ cells/sperm with the feeders were used for in vitro fertilization, normal embryos were obtained. Addition of the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) into culture medium resulted in BrdU-positive sperm and four-cell stage embryos after in vitro fertilization. This culture system should prove useful not only in producing transfected functional sperm, but also in analyzing the regulatory function of testicular somatic cells on the mitosis and meiosis of male germ cells in vertebrates.

The Cancer-Testis Gene, NY-ESO-1, Is Expressed in Normal Fetal and Adult Testes and in Spermatocytic Seminomas and Testicular Carcinoma In Situ

Cancer/testis genes are potential targets for therapeutic genetic and immunologic approaches, and are highly expressed in a large variety of human cancers. However, they are not expressed in normal tissues, with the exception of the testis. The NY-ESO-1 gene is the most recently identified member of the cancer/testis family and its product is one of the most immunogenic tumor antigens. We used immunohistochemistry to investigate the expression of NY-ESO-1 in healthy human prenatal and adult testes and in 59 human testicular tumors of different subtypes. We found that NY-ESO-1 was expressed from 18 weeks until birth in human fetal testes. In the adult testis, NY-ESO-1 was strongly expressed in spermatogonia and in primary spermatocytes, but not in post-meiotic cells or in testicular somatic cells. NY-ESO-1 was not expressed in the Sertoli cells, Leydig cells, classical seminomas, or nonseminomatous germ cells in the 59 testicular tumors. In contrast, NY-ESO-1 was expressed both in carcinomas in situ, which are the earliest stage of testicular tumors (7 of 15 cases), and in spermatocytic seminomas, which are believed to be derived from spermatogonia or primary spermatocytes (8 of 16 cases). We conclude that NY-ESO-1 is a marker that can be used to follow the early progression of testicular tumorigenesis when the tumors present a similar pattern of expression to the cells from which they originated, although the later tumors cease to express NY-ESO-1.

Macrophages and the immune responsiveness of the testis

Immune responses within the testis are regulated in a manner that provides protection for the developing male germ cells, while permitting qualitatively normal inflammatory responses and protection against infection. The large population of resident-type macrophages in the testis is strongly implicated in mediating this specialised immunological environment. Several studies in the rat have shown that testicular macrophages retain their cytotoxic and phagocytic capacity, but have greatly diminished pro-inflammatory function and even exhibit immunosuppressive activity. While the local mechanisms that control the phenotype of the testicular macrophage population are unknown, evidence points to the influence of the testicular somatic cells, the Sertoli and Leydig cells. A smaller but significant population of macrophages that lack expression of resident macrophage markers, is also found in the rat testis. The functional role of these macrophages remains to be defined, but they most likely represent circulating monocytes or newly-arrived testicular macrophages, and, therefore, may contribute to sustaining inflammatory responses within the testis. Further investigation of the immune-related functions of these different macrophage subsets, and the testicular somatic cells, during immunological and inflammatory events should provide a better understanding of how the testicular immune environment is maintained and regulated.

Aromatase expression in male germ cells

The cytochrome P450 aromatase (P450arom) is the terminal enzyme responsible for the formation of estrogens from androgens. According to the age, aromatase activity has been measured in immature and mature rat Leydig cells, as well as in Sertoli cells whereas in pig, ram and human the aromatase is mainly present in Leydig cells. In the rat testis, we have immunolocalised the P450arom not only in Leydig cells but also in germ cells and especially in elongated spermatids. Related to the stage of germ cell maturation, we have shown that the level of P450arom mRNA transcripts decreases, it is much more abundant in younger than in mature germ cells whereas the aromatase activity is two- to four-fold greater in spermatozoa when compared to the two other enriched-germ cell preparations. Moreover, we have reported the existence of alternative splicing events of P450arom mRNA in pachytene spermatocytes and round spermatids giving rise to two isoforms lacking the last coding exon which, therefore, cannot encode functional aromatase molecules. In rat germ cells, the aromatase gene expression is not only under androgen control but also subjected to cytokine (TNFα) and growth factor (TGFβ) regulation. In the bank-vole testis, we have evidenced a synchronisation between a fully developed spermatogenesis and a strong positive immunoreactivity for both P450arom and estrogen receptor (ERβ) in spermatids. Therefore, the aromatase gene expression and its translation in a fully active protein in rodent germ cells evidence an additional site for estrogen production within the testis. Our recent data showing that human ejaculated spermatozoa expressed specific transcripts for P450arom reinforced the observations reported in germ cells of other mammalian species. Together with the widespread distribution of ERs in testicular cells these data bring enlightenments on the hormonal regulation of male reproductive function. Indeed these female hormones (or the ratio androgens/estrogens) do play a physiological role (either directly on germ cells or via testicular somatic cells) in the maintenance of male gonadal functions and obviously, several steps are concerned particularly the spermatid production and the epididymal sperm maturation.

Novel growth factor supporting survival of murine primordial germ cells: evidence from conditioned medium of ter fetal gonadal somatic cells.

The ter (teratoma, chromosome 18) mutation causes a deficiency of primordial germ cells (PGCs) in ter/ter embryos from the ter congenic mouse strain at 8.0 days post coitum (dpc). In order to analyse the function of the ter gene, here we examined effects of conditioned medium (CM) from 14.5 dpc testicular and ovarian somatic cells of +/+, +/ter, or ter/ter genotype on mouse PGCs "mixed-cultured" with own somatic cells on feeder cells. The results showed that +/+ and +/ter CM supported survival in 9.5 and 11.5 dpc ICR PGCs but ter/ter CM did not rescue TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling)-positive apoptosis in the PGCs though it did not affect 5-bromo-2-deoxyuridine incorporation in PGCs. This supportive substance in +/+ CM, not ter/ter CM, was characterized as soluble, heat labile, and larger than 30 kDa. We also found that several known growth factors for PGCs and their receptors were expressed in ter/ter testes as well as +/+ testes, suggesting the ter function is independent. Thus, it was concluded that fetal gonadal somatic cells express a novel PGC growth factor (designated as TER Factor) supporting survival of PGCs not somatic cells and that the PGC deficiency in ter/ter testes is caused by a loss of this factor.

Differential Expression of the Cs and Cα1 Isoforms of the Catalytic Subunit of Cyclic 3′,5′-Adenosine Monophosphate-Dependent Protein Kinase in Testicular Cells1

The amino terminus of the sperm cAMP-dependent protein kinase catalytic subunit (termed C(s)) differs from that of the Calpha1 isoform expressed in most tissues due to the use of alternative transcripts of the Calpha gene. Both Calpha1 and C(s) transcripts are present in testis; C(s) is expressed specifically in spermatogenic cells and is the only C isoform detected in mature sperm. Immunohistochemistry of mouse testis using antibodies specific for C(s) and Calpha1 now shows that Calpha1 is present in somatic testicular cells, spermatogonia, and preleptotene spermatocytes but not in cells that are in later stages of spermatogenesis. In contrast, C(s) is expressed only in midpachytene and later stage spermatocytes and in spermatids. Therefore, C(s) and Calpha1 expression do not overlap. Immunofluorescence microscopic localization of C(s) in murine and ovine sperm reveals that C(s) is located primarily in sperm tail components, including the midpiece mitochondria and the axoneme. Quantitative analysis of Western blots indicates that individual ovine sperm contain approximately 4 x 10(5) molecules of C(s), a seemingly large number for a protein that acts catalytically.

Cellular distribution of translationally controlled tumor protein in rat and human testes

In a recent proteomic study we identified 53 spermatogonial proteins among which was the translationally controlled tumor protein (TCTP). This is a protein previously reported as being implicated in proliferation events in normal and tumoral tissues that had never previously been seen in the testis. The present study was aimed at establishing the complete cellular distribution of TCTP and its transcript and the ontogenetic expression of this gene within the testis. Using an immunohistochemistry technique, an intense TCTP signal was detected in gonocytes (the prespermatogonia) in the fetal rat testis and in spermatogonia within adult human and neonatal and adult rat testes. Meiotic spermatocytes and postmeiotic haploid spermatids were also strongly immunostained in a stage-dependent manner in human and rat testes. In addition, different levels of TCTP expression were also observed in the testicular somatic cells, with strong expression in Leydig cells and peritubular cells, and weak expression in Sertoli cells. Western and Northern blot analyses confirmed the presence of TCTP at all ages studied, with higher levels of RNA expression at 9 and 20 d postpartum, when spermatogonia and primary spermatocytes represent the highest proportion of germ cells: it was also confirmed that TCTP is present in all populations of isolated testicular cells. A transcript of 0.85 kb corresponding to TCTP, was expressed at all ages studied. This transcript was found to be expressed strongly in spermatogonia, somewhat less in isolated Leydig, resident macrophage, peritubular and Sertoli cells, weakly in the primary spermatocytes but not at all in spermatids. Interestingly, in the latter, a different transcript of 1.1 kb was present. The same 1.1 kb transcript appeared in testis extracts from 35 days postpartum onwards, corresponding to an age when spermatids accumulate within the tubules. Of note is that resident macrophages were found to express both the 0.85 and the 1.1 kb transcripts. We conclude that the strong expression of TCTP in spermatogonia makes it highly likely that the protein plays a significant role in spermatogenesis.

Cellular distribution of translationally controlled tumor protein in rat and human testes.

In a recent proteomic study we identified 53 spermatogonial proteins among which was the translationally controlled tumor protein (TCTP). This is a protein previously reported as being implicated in proliferation events in normal and tumoral tissues that had never previously been seen in the testis. The present study was aimed at establishing the complete cellular distribution of TCTP and its transcript and the ontogenetic expression of this gene within the testis. Using an immunohistochemistry technique, an intense TCTP signal was detected in gonocytes (the prespermatogonia) in the fetal rat testis and in spermatogonia within adult human and neonatal and adult rat testes. Meiotic spermatocytes and postmeiotic haploid spermatids were also strongly immunostained in a stage-dependent manner in human and rat testes. In addition, different levels of TCTP expression were also observed in the testicular somatic cells, with strong expression in Leydig cells and peritubular cells, and weak expression in Sertoli cells. Western and Northern blot analyses confirmed the presence of TCTP at all ages studied, with higher levels of RNA expression at 9 and 20 d postpartum, when spermatogonia and primary spermatocytes represent the highest proportion of germ cells: it was also confirmed that TCTP is present in all populations of isolated testicular cells. A transcript of 0.85 kb corresponding to TCTP, was expressed at all ages studied. This transcript was found to be expressed strongly in spermatogonia, somewhat less in isolated Leydig, resident macrophage, peritubular and Sertoli cells, weakly in the primary spermatocytes but not at all in spermatids. Interestingly, in the latter, a different transcript of 1.1 kb was present. The same 1.1 kb transcript appeared in testis extracts from 35 days postpartum onwards, corresponding to an age when spermatids accumulate within the tubules. Of note is that resident macrophages were found to express both the 0.85 and the 1.1 kb transcripts. We conclude that the strong expression of TCTP in spermatogonia makes it highly likely that the protein plays a significant role in spermatogenesis.

Photoperiod-dependent capability of androgen aromatization and the role of estrogens in the bank vole testis visualized by means of immunohistochemistry

Detection of steroid hormone receptors within a target tissue is important for an understanding of their crucial role in regulating of steroids’ action. In the light of recent knowledge on the role of estrogens in male gonads the efforts were undertaken to clarify and discuss a role of androgen receptors, aromatase and estrogen receptors (ER) in mediating testosterone and/or estradiol action in testicular cells of bank voles that were kept under short or long light cycles. Immunohistochemistry was performed on paraplast embedded sections of the bank vole testes. First, androgen receptors were immunolocalized in testicular somatic cells while germ cell did not express any immunoreaction. Moreover, the ability to convert androgens to estrogens by various testicular cells was documented; aromatase immunoexpression was found in testis sections, not only in Leydig cells and Sertoli cells but also in germ cells. Finally, the expression of estrogen receptor-α (ERα) was observed in Leydig cells whereas the presence of estrogen receptor-β (ERβ) was detected in Sertoli and germ cells, namely spermatocytes and spermatids. The cellular distribution of androgen receptors appeared to be light -and age-dependent in adults; immunoexpression of aromatase and ERβ was found to be both age -and photoperiod-dependent in germ cells.

Expression of Apg-1, a member of the Hsp110 family, in the human testis and sperm.

Apg-1 encodes a heat shock protein belonging to the Hsp110 family and is inducible by a 32 degrees C to 39 degrees C heat shock in somatic cells. In mouse testicular germ cells Apg-1 mRNA is constitutively expressed depending on the developmental stage. As human Apg-1 has recently been identified, the expression of Apg-1 in the human testis and sperm was investigated. Expression and heat-inducibility of Apg-1 in the human testicular germ cell tumor cell line, NEC8, was analyzed. Using an antimouse Apg-1 antibody, expression of Apg-1 in the human testis and sperm was examined by western blotting after confirmation of the specificity of the antibody. The cells expressing Apg-1 in the testis were further determined by immunohistochemistry. Slight induction of Apg-1 mRNA was detected in NEC8 cells after 32 degrees C to 39 degrees C temperature shift. In the human testis, the antibody specifically recognized Apg-1, which was absent in the testis without germ cells (Sertoli-cell-only syndrome) or arrested at spermatogonia. Spermatocytes and spermatids, but not testicular somatic cells, were positively stained with the anti-Apg-1 antibody. By western blot analysis, Apg-1 was detected in the preparation enriched for sperm from normal volunteers and infertile patients, but not from azoospermia patients. Apg-1 is developmentally expressed in human testicular germ cells and sperm, suggesting its role in spermatogenesis and fertilization. Identification of substrates for Apg-1 chaperone activity will help elucidate its function.

The road to ovulation: the role of oestrogens

Oestrogens have been known for many years to have a direct influence on folliculogenesis. Oestradiol-17beta (E2) and its analogues have both proliferative and differentiative effects on somatic cells of follicles. Nevertheless, definitive proof of an obligatory role for oestrogen in folliculogenesis and elucidation of the mechanisms subserving its different actions in follicular cells remains elusive. Several recent developments permit a re-examination of the roles and actions of E2 in the follicle. They are: (i) the discovery of a second form of the oestrogen receptor, ERbeta; (ii) the advent of genetically modified mice with deletions in the ERalpha (alphaERKO) ERbeta (BERKO) and the double ER deletions (alphabetaERKO); and (iii) a mouse model of oestrogen deficiency (ArKO) by targeted disruption of the cyp 19 gene encoding the aromatase enzyme. Recent information derived from these models is reviewed to re-assess the roles and actions of oestrogens in follicular dynamics and the phenotypic differentiation of ovarian somatic cells in the ovary. The data demonstrate that oestrogen is obligatory for normal folliculogenesis and that the phenotype of the ovarian somatic cells depends on the steroid milieu. The ArKO mouse provides a model to test the roles of the respective ERs in proliferation and differentiation using specific agonists and antagonists, and to study regulation of the differentiation of ovarian and testicular somatic cells.

Differentiation of mouse primordial germ cells into female or male germ cells.

Mouse primordial germ cells (PGCs) migrate from the base of the allantois to the genital ridge. They proliferate both during migration and after their arrival, until initiation of the sex-differentiation of fetal gonads. Then, PGCs enter into the prophase of the first meiotic division in the ovary to become oocytes, while those in the testis become mitotically arrested to become prospermatogonia. Growth regulation of mouse PGCs has been studied by culturing them on feeder cells. They show a limited period of proliferation in vitro and go into growth arrest, which is in good correlation with their developmental changes in vivo. However, in the presence of multiple growth signals, PGCs can restart rapid proliferation and transform into pluripotent embryonic germ (EG) cells. Observation of ectopic germ cells and studies of reaggregate cultures suggested that both male and female PGCs show cell-autonomous entry into meiosis and differentiation into oocytes if they were set apart from the male gonadal environments. Recently, we developed a two-dimensional dispersed culture system in which we can examine transition from the mitotic PGCs into the leptotene stage of the first meiotic division. Such entry into meiosis seems to be programmed in PGCs before reaching the genital ridges and unless it is inhibited by putative signals from the testicular somatic cells.

Transforming growth factor-beta2 mediates mesenchymal-epithelial interactions of testicular somatic cells.

Transforming growth factor-beta2 (TGFbeta2) is an important mediator of growth and differentiation. We here describe for the first time the complete sequence of the TGFbeta2 complementary DNA derived from peritubular myoid cells of the rat testis. The size of the rat TGFbeta2 complementary DNA was 1245 bp, and the deduced protein sequence contained 414 amino acids. Sequence comparison with the human and mouse amino acid sequences demonstrated 96.4% and 97.9% sequence identities, respectively. To elucidate the functional role of TGFbeta2 in testicular somatic cells, we studied its secretion in vitro in monocultures and cocultures of mesenchymal peritubular and epithelial Sertoli cells. The highest amounts of TGFbeta2 protein were secreted in the cocultures and by peritubular cells, whereas Sertoli cells secreted only minor amounts. Stimulation experiments with FSH revealed a reduced secretion of TGFbeta2 in cocultures, probably mediated by a paracrine interaction of the FSH-responsive Sertoli cells. In contrast, TGFbeta2 secretion by peritubular cells was increased after stimulation with glucocorticoids and after addition of recombinant TGFbeta2, indicating an autoregulation of TGFbeta2. Furthermore, application of recombinant TGFbeta2 to cocultures resulted in an enhanced aggregation and cell clustering of Sertoli cells, pointing to an important role of TGFbeta2 in the paracrine interaction of peritubular and Sertoli cells of the developing rat testis.