Gonocyte Proliferation Research Articles

Developmental Expression of the Translocator Protein 18 kDa (TSPO) in Testicular Germ Cells

Translocator protein (TSPO) is a high affinity 18 kDa drug- and cholesterol-binding protein strongly expressed in steroidogenic tissues where it mediates cholesterol transport into mitochondria and steroid formation. Testosterone formation by Leydig cells in the testis is critical for the regulation of spermatogenesis and male fertility. Male germ cell development comprises two main phases, the pre-spermatogenesis phase occurring from fetal life to infancy and leading to spermatogonial stem cell (SSC) formation, and spermatogenesis, which consists of repetitive cycles of germ cell mitosis, meiosis and differentiation, starting with SSC differentiation and ending with spermiogenesis and spermatozoa formation. Little is known about the molecular mechanisms controlling the progression from one germ cell phenotype to the next. Here, we report that testicular germ cells express TSPO from neonatal to adult phases, although at lower levels than Leydig cells. TSPO mRNA and protein were found at specific steps of germ cell development. In fetal and neonatal gonocytes, the precursors of SSCs, TSPO appears to be mainly nuclear. In the prepubertal testis, TSPO is present in pachytene spermatocytes and dividing spermatogonia. In adult testes, it is found in a stage-dependent manner in pachytene spermatocyte and round spermatid nuclei, and in mitotic spermatogonia. In search of TSPO function, the TSPO drug ligand PK 11195 was added to isolated gonocytes with or without the proliferative factors PDGF and 17β-estradiol, and was found to have no effect on gonocyte proliferation. However, TSPO strong expression in dividing spermatogonia suggests that it might play a role in spermatogonial mitosis. Taken together, these results suggest that TSPO plays a role in specific phases of germ cell development.

Zebrafish monosex population reveals female dominance in sex determination and earliest events of gonad differentiation

The zebrafish is a popular model for genetic analysis and its sex differentiation has been the focus of attention for breeding purposes. Despite numerous efforts, very little is known about the mechanism of zebrafish sex determination. The lack of discernible sex chromosomes and the difficulty of distinguishing the sex of juvenile fish are two major obstacles that hamper the progress in such studies. To alleviate these problems, we have developed a scheme involving methyltestosterone treatment followed by natural mating to generate fish with predictable sex trait. Female F1 fish that gave rise to all-female offspring were generated. This predictable sex trait enables characterization of gonadal development in juvenile fish by histological examination and gene expression analysis. We found the first sign of zebrafish sex differentiation to be ovarian gonocyte proliferation and differentiation at 10 to 12 days post-fertilization (dpf). Somatic genes were expressed indifferently at 10 to 17 dpf, and then became sexually dimorphic at three weeks. This result indicates clear distinction of male and female gonads derived independently from primordial gonads. We classified the earliest stages of zebrafish sex determination into the initial preparation followed by female germ cell growth, oocyte differentiation, and somatic differentiation. Our genetic selection scheme matches the prediction that female-dominant genetic factors are required to determine zebrafish sex.

TGFβ signaling in male germ cells regulates gonocyte quiescence and fertility in mice

During testis development, proliferation and death of gonocytes are highly regulated to establish a standard population of adult stem spermatogonia that maintain normal spermatogenesis. As Transforming Growth Factor beta (TGFbeta) can regulate proliferation and apoptosis, we investigated its expression and functions during testis development. We show that TGFbeta2 is only expressed in quiescent gonocytes and decreases gonocyte proliferation in vitro. To study the functions of TGFbeta2, we developed conditional mice that invalidate the TGFbeta receptor type II in germ cells. Most of the knock-out animals die during fetal life, but the surviving adults show a reduced pool of spermatogonial stem/progenitor cells and become sterile with time. Using an organ culture system mimicking in vivo development, we show higher proportions of proliferating and apoptotic gonocytes from 13.5 dpc until 1 dpp, suggesting a reduction of germinal quiescence in these animals. Conversely, a 24-hour TGFbeta2-treatment of explanted wild-type testes, isolated every day from 13.5 dpc until 1 dpp, increased the duration of quiescence. These data show that the TGFbeta signaling pathway plays a physiological role during testis development by acting directly as a negative regulator of the fetal and neonatal germ cell proliferation, and indicate that the TGFbeta signaling pathway might regulate the duration of germ cell quiescence and is necessary to maintain adult spermatogenesis.

Neonatal Gonocyte Differentiation in Mongolian GerbilMeriones unguiculatusInvolves Asynchronous Maturation of Seminiferous Cords and Rapid Formation of Transitional Cell Stage

This study describes the neonatal differentiation of the Mongolian gerbil gonocytes, focusing on the relationship between its relocation to the basement membrane, apoptosis and postrelocation changes and also the distribution of androgen receptors (AR). Testes of gerbils from 1 to 35 days of age (d) were examined by high resolution light microscopy and immunocytochemistry for proteins PCNA, VASA, and AR as well as by the TUNEL method. Gonocytes were quantified according to degree of relocation into nonrelocated, relocating and relocated. Most of them were found in the center of seminiferous cords at 1 d but a small number of relocating and relocated gonocytes were already visible in the first postnatal day. After relocation, gonocytes change phenotypically to a transitional stage designated herein prospermatogonia. Both gonocyte relocation and transformation into spermatogonial lineage occur asynchronously in the seminiferous cords, mainly after 7 d. Gonocyte proliferation began before but peak after their relocation to basement membrane at the prospermatogonia stage. Higher levels of gonocyte apoptosis were found at 7 d and 21 d. From this time onward gonocytes were not found. Gonocytes and prospermatogonia showed high amounts of AR in their cytoplasm contrary to spermatogonial subtypes, indicating a possible AR inactivation in these cells. In conclusion, the process of gonocyte relocation in the gerbil extends until the second postnatal week, leads to their rapid differentiation into prospermatogonia and occurs simultaneously with the loss of androgen sensitivity. Differently from other laboratory rodents, the events regarding gonocyte maturation in the gerbil last longer and occur asynchronously in seminiferous cords.

Interdependence of Platelet-Derived Growth Factor and Estrogen-Signaling Pathways in Inducing Neonatal Rat Testicular Gonocytes Proliferation1

We previously found that platelet-derived growth factor (PDGF) and 17beta-estradiol stimulate gonocyte proliferation in a dose-dependent, nonadditive manner. In the present study, we report that gonocytes express RAF1, MAP2K1, and MAPK1/3. Inhibition of RAF1 and MAP2K1/2, but not phosphoinositide-3-kinase, blocked PDGF-induced proliferation. AG-370, an inhibitor of PDGF receptor kinase activity, suppressed not only PDGF-induced proliferation but also that induced by 17beta-estradiol. In addition, RAF1 and MAP2K1/2 inhibitors blocked 17beta-estradiol-activated proliferation. The estrogen receptor antagonist ICI 182780 inhibited both the effects of 17beta-estradiol and PDGF. PDGF lost its stimulatory effect when steroid-depleted serum or no serum was used. Similarly, 17beta-estradiol did not induce gonocyte proliferation in the absence of PDGF. The xenoestrogens genistein, bisphenol A, and DES, but not coumestrol, stimulated gonocyte proliferation in a dose-dependent and PDGF-dependent manner similarly to 17beta-estradiol. Their effects were blocked by ICI 182780, suggesting that they act via the estrogen receptor. AG-370 blocked genistein and bisphenol A effects, demonstrating their requirement of PDGF receptor activation in a manner similar to 17beta-estradiol. These results demonstrate the interdependence of PDGF and estrogen pathways in stimulating in vitro gonocyte proliferation, suggesting that this critical step in gonocyte development might be regulated in vivo by the coordinated action of PDGF and estrogen. Thus, the inappropriate exposure of gonocytes to xenoestrogens might disrupt the crosstalk between the two pathways and potentially interfere with gonocyte development.

In vitro Culture of Testicular and Ovarian Gonocytes Obtained from 19-day Incubated Chicken Embryos and Subsequent Colonization into Gonads of Recipient Embryos

Testicular gonocytes differentiate into spermatogonia and spermatogonial stem cells could be established by culturing spermatogonia in vitro. Spermatogonial stem cells differentiate exclusively into spermatozoa and thus they are very useful for genetic manipulation in chickens. The present study was carried out to examine the possibility of in vitro proliferation of testicular and ovarian gonocytes in chickens. Gonads were obtained from 19-day incubated chicken embryos and the dissociated gonadal cells were cultured in vitro. Then, non-adherent cells containing gonocytes were collected and cultured further. It was confirmed that a part of the testicular gonocytes proliferated in vitro, when analyzed by anti-CVH antibody. The proliferated testicular gonocytes occasionally formed cell colonies. The cultured testicular gonocytes were successfully implanted in the gonads by transferring them into the coelomic epithelium that corresponds to the future gonadal region of recipient embryos. However, no apparent proliferation was observed in the ovarian gonocytes in vitro. These results suggest that testicular goncytes can be cultured in vitro and are one of the candidate cells for genetic manipulation in chickens.

Time- and Dose-Related Effects of Di-(2-ethylhexyl) Phthalate and Its Main Metabolites on the Function of the Rat Fetal Testis in Vitro

BackgroundEndocrine-disrupting effects of phthalates are understood primarily from in utero exposures within the fetal rat testis. Nevertheless, their path of action, dose–response character, and cellular target(s) within the fetal testis are not known.ObjectivesIn this study we investigated the effects of di-(2-ethylhexyl) phthalate (DEHP), mono-(2-ethylhexyl) phthalate (MEHP), and several of their metabolites on the development of organo-cultured testes from rat fetus.MethodsWe removed testes from 14.5-day-old rat fetuses and cultured them for 1–3 days with or without DEHP, MEHP, and the metabolites.ResultsDEHP (10−5 M) produced a proandrogenic effect after 3 days of culture, whereas MEHP disrupted testis morphology and function. Leydig cells were the first affected by MEHP, with a number of them being inappropriately located within some seminiferous tubules. Additionally, we found a time- and dose-dependent reduction of testosterone. By 48 hr, gonocyte proliferation had decreased, whereas apoptosis increased. Sertoli cell number was unaffected, although some cells appeared vacuolated, and production of anti-Müllerian hormone decreased in a time- and dose-dependent manner. The derived metabolite mono-(2-ethyl-5-hydroxyhexyl) phthalate was the only one to cause deleterious effects to the rat fetal testis in vitro.ConclusionWe hope that this in vitro method will facilitate the study of different phthalate esters and other endocrine disruptors for direct testicular effects.

Platelet-Derived Growth Factor Receptor β-Subtype Regulates Proliferation and Migration of Gonocytes

Proliferation and migration of gonocytes, the precursors of spermatogonial stem cells, to the germline niche in the basal membrane of the seminiferous tubules, are two crucial events that take place between postnatal d 0.5 (P0.5) and P5.0 in the mouse and involve a selection of the cells that are committed to the germline stem cells lineage. Here we show that from embryonic d 18.0 (E18) and up to P5, the gonocytes express platelet-derived growth factor (PDGF) receptor beta-subtype (PDGFR-beta) and that during the same time period, the Sertoli cells express PDGF-B and PDGF-D, both ligands for PDGFR-beta. Inhibition of the PDGFR-beta tyrosine kinase activity during the first five postnatal days provokes a profound reduction of gonocyte number through inhibition of their proliferation and induction of apoptosis. Moreover, we found that PDGFR-beta ligands are chemotactic for gonocytes. These data suggest that PDGFR-beta activation has the remarkable capability to drive the selection, survival, and migration of the gonocytes from the center of the seminiferous tubules to the testicular germline niche on the basal membrane.

233. A role for activin/inhibin in mouse gonocyte relocation and proliferation

Gonocytes in the testis resume proliferation after birth and relocate to contact the basement membrane of the seminiferous cords where they become spermatogonia. A previous in vitro study indicated that activin can increase gonocyte numbers on day 3 postpartum (dpp) rat testis, while the activin antagonist, follistatin, together with FSH, increased the number of spermatogonia (Meehan et al. 2000). The aim of this study was to understand how FSH, activin and inhibin, a potent activin antagonist, interact to influence gonocyte proliferation and relocation in the newborn mouse testis using in vivo and in vitro approaches. Two mouse models were analysed, the inhibin α knock out (inh a −/−) mouse and the InhbaBK mouse. The Inh a −/− mouse lacks inhibin, and thus activin acts unopposed by its most potent antagonist (Matzuk et al. 1992). The InhbaBK mouse has the Inhbb allele inserted into the Inhba locus, thus directing the expression of the less bioactive activin βB, in the spatiotemporal pattern of activin βA (Brown et al. 2000). In addition, an in vitro model was developed in which 1dpp wild type testis fragments were cultured in hanging drops for 24 h with the addition of combinations of activin, inhibin and FSH. Gonocyte proliferation in inh a −/− was assessed using proliferating cell nuclear antigen (PCNA). A significant increase in germ cell proliferation and relocation to the basement membrane was measured in 0dpp inh a −/−, while no difference was observed at 4dpp. The opposite was observed in InhbaBK mice, with reduced gonocyte migration in mutant animals at 0dpp. In vitro, inhibin seemed to inhibit proliferation and reduce the percentage of relocated gonocytes while FSH showed a tendency for the opposite effect on gonocyte migration. These findings show that inhibin levels affect germ cell development during early postnatal development in mouse testis influencing both cell maturation and proliferation. (1) Meehan T, Schlatt S, O'Bryan MK, de Kretser DM, Loveland KL. Regulation of germ cell and Sertoli cell development by activin, follistatin, and FSH. Dev Biol. 2000 Apr 15;220(2):225–37. (2) Matzuk MM, Finegold MJ, Su JG, Hsueh AJ, Bradley A. Alpha-inhibin is a tumour-suppressor gene with gonadal specificity in mice. Nature. 1992 Nov 26;360(6402):313–9. (3) Brown CW, Houston-Hawkins DE, Woodruff TK, Matzuk MM. Insertion of Inhbb into the Inhba locus rescues the Inhba null phenotype and reveals new activin functions. Nat Genet. 2000 Aug;25(4):453–7.

Male fetal germ cells are targets for androgens that physiologically inhibit their proliferation

In adulthood, the action of androgens on seminiferous tubules is essential for full quantitatively normal spermatogenesis and fertility. In contrast, their role in the fetal testis, and particularly in fetal germ cell development, remains largely unknown. Using testicular feminized (Tfm) mice, we investigated the effects of a lack of functional androgen receptor (AR) on fetal germ cells, also named gonocytes. We demonstrated that endogenous androgens/AR physiologically control normal gonocyte proliferation. We observed an increase in the number of gonocytes at 17.5 days postconception resulting from an increase in proliferative activity in Tfm mice. In a reciprocal manner, gonocyte proliferation is decreased by the addition of DHT in fetal testis organotypic culture. Furthermore, the AR coregulator Hsp90alpha (mRNA and protein) specifically expressed in gonocytes was down-regulated in Tfm mice at 15.5 days postconception. To investigate whether these effects could result from direct action of androgens on gonocytes, we collected pure gonocyte preparations and detected AR transcripts therein. We used an original model harboring a reporter gene that specifically reflects AR activity by androgens and clearly demonstrated the presence of a functional AR protein in fetal germ cells. These data provide in vivo and in vitro evidence of a new control of endogenous androgens on gonocytes identified as direct target cells for androgens. Finally, our results focus on a new pathway in the fetal testis during the embryonic period, which is the most sensitive to antiandrogenic endocrine disruptors.

Maintenance of spermatogenesis requires TAF4b, a gonad-specific subunit of TFIID

The establishment and maintenance of spermatogenesis in mammals requires specialized networks of gene expression programs in the testis. The gonad-specific TAF4b component of TFIID (formerly TAF(II)105) is a transcriptional regulator enriched in the mouse testis. Herein we show that TAF4b is required for maintenance of spermatogenesis in the mouse. While young Taf4b-null males are initially fertile, Taf4b-null males become infertile by 3 mo of age and eventually exhibit seminiferous tubules devoid of germ cells. At birth, testes of Taf4b-null males appear histologically normal; however, at post-natal day 3 gonocyte proliferation is impaired and expression of spermatogonial stem cell markers c-Ret, Plzf, and Stra8 is reduced. Together, these data indicate that TAF4b is required for the precise expression of gene products essential for germ cell proliferation and suggest that TAF4b may be required for the regulation of spermatogonial stem cell specification and proliferation that is obligatory for normal spermatogenic maintenance in the adult.

Early testicular effects in rats perinatally exposed to DEHP in combination with DEHA—apoptosis assessment and immunohistochemical studies

This study aimed to characterize the effects of di(2-ethylhexyl) phthalate (DEHP) on the fetal rat testes and relate them to the effects seen in adults. Histopathological effects in fetal testes were examined with immunohistochemistry for anti-Müllerian hormone (AMH), 3β-hydroxysteroid dehydrogenase, smooth muscle actin (SMA), proliferating cell nuclear antigen (PCNA), histone H3 and vimentin. Additionally, testicular apoptosis levels were assessed in fetal, prepubertal and adult rats. As the plasticizer di(2-ethylhexyl) adipate (DEHA) has similarities with DEHP in chemical structure and metabolism, we investigated if the testicular effects of DEHP were modulated by co-administration with DEHA. Wistar rats were gavaged during gestation and lactation with vehicle, DEHP (300 or 750 mg/kg/day), or DEHP (750 mg/kg/day) in combination with DEHA (400 mg/kg/day), and male offspring were examined at gestation day (GD) 21, postnatal day (PND) 22, 26 and 190. In fetal testes, Leydig cells were found in large clusters containing AMH positive Sertoli cells. At GD 21, seminiferous chords appeared enlarged with an apparently increased number of gonocytes. However, proliferation of gonocytes did not appear increased. A few animals had a high number of TUNEL positive apoptotic cells in degenerating seminiferous tubules at PND 22 and 190, whereas most exposed animals had low levels of germ cell apoptosis at GD 21, PND 22 or PND 26, as evaluated by DNA laddering, TUNEL staining, Caspase-3 immunohistochemistry and Caspase-3 activity measurement. No differences between DEHP and DEHP + DEHA exposed groups were observed.

An Overview of Cell Renewal in the Testis Throughout the Reproductive Cycle of a Seasonal Breeding Teleost, the Gilthead Seabream (Sparus aurata L)1

The gilthead seabream is a protandrous hermaphrodite seasonal breeding teleost with a bisexual gonad that offers an interesting model for studying the testicular regression process that occurs in both seasonal testicular involution and sex change. Insofar as fish reproduction is concerned, little is known about cell renewal and elimination during the reproductive cycle of seasonal breeding teleosts with asynchronous spermatogenesis. We have previously described how acidophilic granulocytes infiltrate the testis during postspawning where, surprisingly, they produce interleukin-1beta, a known growth factor for mammalian spermatogonia, rather than being directly involved in the elimination of degenerative germ cells. In this study, we are able to discriminate between spermatogonia stem cells and primary spermatogonia according to their nuclear and cytoplasmic diameters and location in the germinal epithelium, finding that these two cell types, together with Sertoli cells, proliferate throughout the reproductive cycle with a rate that depends on the reproductive stage. Thus, during spermatogenesis the spermatogonia stem cells, the Sertoli cells, and the developing germ cells (primary spermatogonia, A and B spermatogonia, and spermatocytes) in the germinal compartment, and cells with fibroblast-shaped nuclei in the interstitial tissue proliferate. However, during spawning, the testis shows few proliferating cells. During postspawning, the resumption of proliferation, the occurrence of apoptotic spermatogonia, and the phagocytosis of nonshed spermatozoa by Sertoli cells lead to a reorganization of both the germinal compartment and the interstitial tissue. Finally, the proliferation of spermatogonia increases during resting when, unexpectedly, both oogonia and oocytes also proliferate. This proliferative pattern was correlated with the gonadosomatic index, testicular morphology, and testicular and gonad areas, suggesting that complex mechanisms operate in the regulation of gonocyte proliferation in hermaphrodite fish.

Time- and dose-related effects of estradiol and diethylstilbestrol on the morphology and function of the fetal rat testis in culture.

The mechanisms underlying the action of estrogens in the fetal testis are still largely obscure. In particular, whether this action is direct or indirect remains largely unexplored. This study was aimed at investigating the effect of estradiol (E2) and diethylstilbestrol (DES) on the testis from 14.5-day-old rat fetuses in culture, at concentrations ranging from 4 x 10(-10) M (Kd of E2 for the estrogen receptors [ER]: 1-4 x 10(-10) M) to 4 x 10(-6) M (concentration previously shown in the literature to affect in vitro gonocyte proliferation). Exposure to DES and E2 decreased gonocyte number, the effects of DES being much more drastic than those of E2. Gonocyte number decreased in a concentration-dependent manner (day 3: -5%, -16%, and -80% at 4 x 10(-10) M, 4 x 10(-8) M, and 4 x 10(-6)M of DES, respectively), as well as in a time-dependent manner (at 4 x 10(-6) M DES: -31% on day 1, -60% on day 2, and -80% on day 3). This was due to a decrease in the gonocyte mitotic index and a dramatic increase in apoptosis. Importantly, in the presence of the anti-estrogen ICI 182.780 (ICI), the effect of DES was abolished. Sertoli cell number subsequently decreased (day 3), although the rate of apoptosis did not increase. These changes were less dramatic than those observed with gonocytes and were due to a decrease in Sertoli cell proliferation, which was not antagonized by ICI. Addition of 4 x 10(-6) M DES had no effect on basal Sertoli cell cyclic adenosine 5'-monophosphate (cAMP) levels or on follicle-stimulating hormone (FSH)-stimulated cAMP production after adjustment for Sertoli cell number. Finally, estrogens reduced both Leydig cell number (-26% on day 3, 4 x 10(-6) M DES) and basal and luteinizing hormone (LH)-stimulated testosterone production. The latter effects were antagonized by ICI. 1) E2 and DES induce alterations in the germ line and in somatic cells; 2) gonocyte alteration was the first event detected, and the action of estrogens at this level was mediated by ER, as is the case in Leydig cells; and 3) these data should help us to understand estrogen effects on the fetus and should be considered in the context of the debate on environmental estrogens.

Prenatal Exposure to Estrogenic Compounds Alters the Expression Pattern of Platelet-Derived Growth Factor Receptors α and β in Neonatal Rat Testis: Identification of Gonocytes as Targets of Estrogen Exposure1

We examined the effects of maternal exposure to estrogens on platelet-derived growth factor (PDGF) receptor (PDGFR) expression in newborn rat testis. Pregnant rats were treated from gestation Day 14 to birth with corn oil containing diethylstilbestrol, bisphenol A, genistein, or coumestrol by gavage or subcutaneous injection. These treatments induced a dose-dependent increase in the expression of PDGFR alpha and beta mRNAs, determined by semiquantitative reverse transcription polymerase chain reaction, though diethylstilbestrol had a biphasic effect on both mRNAs. In situ hybridization analysis showed that PDGFRalpha mRNA increased mostly in the interstitium, while PDGFRbeta mRNA increased both in the interstitium and seminiferous cords. Immunohistochemical studies of PDGFRalpha and beta proteins revealed that both receptors were present in testis before and after birth and that they were upregulated upon treatment with estrogens in 3-day-old rats, with PDGFRbeta increasing dramatically in gonocytes. PDGFRalpha and beta mRNAs and proteins were also found in purified gonocytes. Our previous finding that PDGF and 17beta-estradiol induce gonocyte proliferation in vitro, together with the present finding that in vivo exposure to estrogens upregulates PDGF receptors in testis, suggest that PDGF pathway is a target of estrogens in testis. In addition, these data identify PDGFRbeta in gonocytes as a major target of gestational estrogen exposure, suggesting that estrogen may have a physiological interaction with PDGF during gonocyte development. These results, however, do not exclude the possibility that the effects of the compounds examined in this study might be due to estrogen receptor-independent action(s).

Fetal testosterone insufficiency and abnormal proliferation of Leydig cells and gonocytes in rats exposed to di(n-butyl) phthalate

Adult male rats previously exposed on gestation days (GD) 12–21 to di( n-butyl) phthalate (DBP) have reproductive tract malformations, particularly agenesis of the epididymis, decreased sperm production, and Leydig cell hyperplasia and adenomas. Although similar effects are produced by the potent androgen receptor (AR) antagonist flutamide and are indicative of disruption of male sexual differentiation via an antiandrogenic mechanism, DBP is not an AR antagonist. The purpose of the study was to determine whether DBP causes pathologic changes and alterations in androgen status in the testis during the prenatal period of male reproductive tract differentiation. Pregnant CD rats were given corn oil, DBP (500 mg/kg/day), or flutamide (100 mg/kg/day) p.o. on GD 12–21. At GD 16–21, DBP caused hyperplasia of Leydig cells, many of which were 3β-hydroxysteroid dehydrogenase- and/or AR-positive. Focal areas of hyperplasia had increased numbers of Leydig cells positive for proliferating cell nuclear antigen (PCNA). At GD 21, testis atrophy was apparent, seminiferous cords in DBP-exposed fetuses were enlarged and contained multinucleated gonocytes that, unlike controls, were PCNA-positive. DBP, but not flutamide, markedly decreased testicular testosterone levels at GD 18 and 21. Fewer epididymal ducts and reduced AR staining in some ducts were evident with DBP treatment, whereas decreased overall AR staining was seen with flutamide in the presence of mild Leydig cell hyperplasia. Leydig cell proliferation is likely a compensatory mechanism to increase testicular steroidogenesis triggered by testosterone insufficiency. The overall decrease in androgen concentration is not corrected and results in reproductive tract malformations. The multinuclearity and proliferation of gonocytes suggests an underlying Sertoli cell dysfunction.

Study of the gonocyte cell cycle in irradiated TP53 knockout mouse foetuses and newborns

Purpose : To investigate the role of p53 in gonocyte cell-cycle arrest in rodents with or without radiation exposure. Materials and methods : Pregnant p53 (+/-) mice, mated with p53 (-/-) males, were exposed to 137 Cs γ-rays at day 18 postcoitum (p.c.) at doses ranging from 1.5 Gy to 3 Gy. The gonocyte cell cycle was studied in p53 (-/-) male foetuses and newborns after BrdU incorporation and DAPI staining, and compared with those of p53 wild-type animals. Results : The proliferation of gonocytes in wild-type mice is normally arrested between day 16 p.c. and birth, a period when p53 is strongly expressed in gonocytes; p53 high expression is prolonged in all gonocytes after 3 Gy irradiation. In p53 (-/-) mice, this period of gonocyte cell-cycle arrest is not modified, compared with wild-type mice. It is also prolonged after a 3 Gy exposure. Conclusion : Two hypotheses are proposed. Either p53 is not involved in the control of gonocyte cell-cycle arrest in control and irradiated mice, or its role is redundant in this process.

Growth factor and somatic cell regulation of mouse gonocyte-derived colony formation in vitro

At birth, the mouse gonocyte does not resume mitotic activity for several days in vivo but, in an in vitro clonogenic system, cell division commences soon after culture. Somatic testis cell underlays had potent inhibitory activity on gonocyte-derived colony formation (23 +/- 15% compared with 84 +/- 1% in controls; P = 0.0001) when added to cultures of gonocytes in vitro. A Sertoli cell line, TM4B, had an even more pronounced effect on gonocyte clonogenic capacity, with 1 +/- 1% compared with 72 +/- 17% colony formation in controls (P = 0.0003). Testis cells appeared to have a direct inhibitory effect since testis-conditioned medium did not show a significant reduction in the number of colonies. The observed reduction in colony formation with the testis cell underlay was not accounted for by decreased attachment of gonocytes as simultaneous addition of a single cell suspension of testis cells was still effective in significantly reducing colony number when compared with controls (P = 0.01). Therefore, the observed inhibition exerted by testis cells appears to be a consequence of decreased proliferation of gonocytes. Growth factors belonging to the transforming growth factor beta superfamily which are known to be expressed in testis, such as transforming growth factor beta and epidermal growth factor, did not exert any inhibitory action on gonocyte-derived colony formation when added together or alone. However, a shift to a smaller colony size occurred in the presence of transforming growth factor beta and transforming growth factor beta plus epidermal growth factor, indicating a reduction in colony cell proliferation. Evidence for the expression of the Mullerian inhibiting substance receptor on newborn gonocytes using in situ hybridization was inconclusive. This finding was in agreement with the lack of a direct action of Mullerian inhibiting substance on the formation of gonocyte-derived colonies in vitro. Leukaemia inhibitory factor, alone or in combination with forskolin, had neither an inhibitory nor an enhancing effect on gonocyte-derived colony formation. An in vitro clonogenic method to assay for the proliferation of gonocytes in the presence of specific growth factors, cell lines, testis cell underlays and cell suspensions was used to identify a somatic cell-mediated inhibitor which may be responsible for the inhibitory action on gonocyte proliferation in vivo shortly after birth.

Neonatal mouse gonocyte proliferation assayed by an in vitro clonogenic method

Survival and proliferation of mouse gonocytes was studied using a single cell clonogenic assay in vitro. The effect of growth factors and extracellular matrix on clonogenic development was quantitated. Fundamental requirements for growth of neonatal gonocytes included addition of fetal calf serum and coating culture wells with collagen IV alone or with added fibronectin. After 4-5 days, colonies ranged in size from four to > 128 cells, and some contained very elongated cells indicating migratory behaviour. Soluble stem cell factor did not have any effect on clonogenicity, although STO (subline of SIM mouse fibroblasts) cells, which produce membrane-bound stem cell factor, reduced colony formation from 79 +/- 5.9% to 20 +/- 3.3% without added growth factor. The majority of gonocytes and type A spermatogonia express the c-kit receptor according to in situ hybridization studies. However, the results indicate that the receptor may not be functional in neonatal gonocytes and their immediate progeny. The current assay for gonocytes can be extended to test new growth factors or proliferation-inducing agents directly, as well as to study cell-cell interactions. This assay and long-term propagation of neonatal germ cells will provide the much needed resources to enable greater understanding of the early development of germ cells.

Regulation of rat testis gonocyte proliferation by platelet-derived growth factor and estradiol: identification of signaling mechanisms involved.

To determine what factors regulate gonocyte proliferation in newborn rats, we first examined the expression of several signal transduction molecules by immunocytochemistry in 3-day-old rat testis sections. We found that gonocytes specifically expressed the iota and zeta isoforms of protein kinase (PK) C (PKC) and the phosphatidylinositol 3-kinase (PI3-K). Because both the zeta PKC and PI 3-K have been shown to play a role in platelet-derived growth factor (PDGF)-induced cell proliferation, we examined the effects of PDGF on gonocytes. For this, we developed a method to obtain highly purified and viable gonocytes in culture. After enzymatic digestion, differential adhesion, and two successive gradient fractionations, the gonocyte suspension obtained was over 90% pure, as assessed by light microscopy. The viability of cultured gonocytes exceeded 90% after 48 h in the presence of 2.5% FBS used as a survival factor. Immunodetection studies showed that isolated gonocytes expressed zeta PKC, PI 3-K, and the PDGF receptor. Treatment with 10 ng/ml PDGF induced a 4-fold increase of bromodeoxyuridine incorporation into gonocytes (from 5% proliferative gonocytes under basal conditions to 20% in the presence of PDGF). Because neonatal Sertoli cells secrete high levels of the growth promoting steroid, 17 beta-estradiol, we also tested its effect and found that it induced gonocyte proliferation at a level comparable with that of PDGF and that this effect was blocked by the estrogen receptor antagonist, ICI 164384. The combination of PDGF and estradiol, however, was not additive, suggesting that their effects were mediated by common molecular target(s). These results demonstrate that PDGF and estradiol activate gonocyte proliferation in vitro, suggesting that they may act as the physiological regulators of gonocyte development in vivo.