Immunolocalization of albumin and transferrin in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis

The localization of albumin and transferrin was examined immunohistochemically in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis. These proteins appeared as early as the 13th day of gestation in migrating primordial germ cells before Sertoli cell differentiation. In the fetal testis, strong immunoreactivity was only detected in the gonocytes. In the prepubertal testis, spermatogonia, primary spermatocytes, and some Sertoli cells accumulate albumin and transferrin. At puberty, different patterns of immunostaining of the germ cells were observed at the various stages of the cycle of the seminiferous epithelium. Diplotene spermatocytes at stage XIII, spermatocytes in division at stage XIV, and round spermatids at stages IV–VIII showed maximal staining. Labeling was evident in the cytoplasm of adult Sertoli cells. Albumin and transferrin staining patterns paralleled each other during ontogenesis.     

Regulation of the proliferation of cocultured gonocytes and Sertoli cells by retinoids,triiodothyronine, and intracellular signaling factors: differences between fetal and neonatal cells

The regulation of early fetal germ cell growth has not been studied in cell culture, probably due to the poor survival of these cells. However, cell culture is the only system in which the control of cell growth can be studied independently of the influence of secreted testicular factors, which are diluted in the medium. We successfully cultured dispersed testicular cells from 16.5-day-old rat fetuses in defined medium and compared the growth of these cells with that of cells from 3-day-old neonates. In this system, fetal gonocytes displayed low levels of mitotic activity and their numbers remained stable. In contrast, neonatal gonocytes displayed high levels of mitotic activity and increased in number, these characteristics resembling those observed in vivo. We found that retinoic acid had deleterious effects on the number of gonocytes but did not affect Sertoli cell proliferation in fetal and neonatal cell cultures. Moreover, in fetal cell cultures, the decrease in the number of gonocytes resulted from a decrease in mitotic activity, probably due to a direct effect of retinoids on fetal gonocytes. Among the selective agonists for the retinoic acid receptor (RARalpha agonist, RARbeta agonist, and RARgamma agonist) and the retinoic X receptor (pan-RXR agonist) tested, only the RARalpha agonist reproduced the effects of retinoic acid at concentrations lower than its Kd value in both fetal and neonatal cell cultures. As both RARalpha and RXRalpha are present in fetal and neonatal gonocytes, we suggest that retinoic acid exerts its effects on gonocytes via a RARalpha-RXRalpha heterodimer, with RARalpha functioning as an active partner and RXRalpha as a passive partner. In this culture system, we show for the first time that triiodothyronine (T3) inhibits testicular fetal Sertoli cell and germ cell growth. We also tested intracellular signaling factors and found that a cAMP analog increased Sertoli cell proliferation and germ cell survival in both fetal and neonatal cells whereas phorbol esters (PMA) strongly inhibited the proliferation of fetal but not of neonatal gonocytes. None of the tested factors (T3, dbcAMP, and PMA) seemed to interact with the all-trans retinoic acid pathway. Thus, fetal gonocytes and neonatal gonocytes differ in intrinsic properties, and their growth is not regulated in the same manner. Despite their low level of mitotic activity, fetal gonocytes were more sensitive to various factors than neonatal gonocytes.     

Regulation of germ cell and Sertoli cell development by activin, follistatin, and FSH

We have demonstrated a role for activin A, follistatin, and FSH in male germ cell differentiation at the time when spermatogonial stem cells and committed spermatogonia first appear in the developing testis. Testis fragments from 3-day-old rats were cultured for 1 or 3 days with various combinations of these factors, incubated with bromodeoxyuridine (BrdU) to label proliferating cells, and then processed for stereological analysis and detection of BrdU incorporation. Gonocyte numbers were significantly elevated in cultures treated with activin, while the combination of FSH and the activin antagonist, follistatin, increased the proportion of spermatogonia in the germ cell population after 3 days. All fragment groups treated with FSH contained a significantly higher proportion of proliferating Sertoli cells, while activin and follistatin each reduced Sertoli cell division. In situ hybridization and immunohistochemistry on normal rat testes demonstrated that gonocytes, but not spermatogonia, contain the activin beta(A) subunit mRNA and protein. In contrast, gonocytes first expressed follistatin mRNA and protein at 3 days after birth, concordant with the transition of gonocytes to spermatogonia. Collectively, these data demonstrate that germ cells have the potential to regulate their own maturation through production of endogenous activin A and follistatin. Sertoli cells were observed to produce the activin/inhibin beta(A) subunit, the inhibin alpha subunit, and follistatin, demonstrating that these cells have the potential to regulate germ cell maturation as well as their own development. These findings indicate that local regulation of activin bioactivity may underpin the coordinated development of germ cells and somatic cells at the onset of spermatogenesis.     

Di(n-butyl) phthalate has no effect on the rat prepubertal testis despite its estrogenic activity in vitro

The aim of this study was to assess the impact of di(n-butyl) phthalate (DBP) on the rat's prepubertal testis. Male Wistar rats were given daily subcutaneous injections with DBP (20 or 200 μg) or a vehicle from the 5th to the 15th postnatal day (pd). On the 16(th) pd, the rats were euthanized, and the testes were dissected, weighed, and paraffin embedded. The blood was collected to determine the serum levels of testosterone (T), estradiol (E) and FSH. The following parameters were assessed in the testis sections: diameter and length of seminiferous tubules (st), numbers of spermatogonia A + intermediate + B (A/In/B), preleptotene spermatocytes (PL), leptotene + zygotene + pachytene spermatocytes (L/Z/PA) and Sertoli cells per testis, percentage of st containing gonocytes or pachytene spermatocytes or lumen. An estrogenicity in vitro test was performed by means of a transgenic yeast strain expressing human estrogen receptor alpha. At both doses, DBP had no influence on testis and seminal vesicle weight, st diameter and length, number of germ and Sertoli cells per testis, percentage of st containing gonocytes or pachytene spermatocytes or lumen. DBP did not change E, T or FSH serum levels. The in vitro yeast screen showed that DBP was a weak estrogenic compound, approximately six to seven orders of magnitude less potent than 17β-estradiol. In conclusion, exposure of a rat to DBP in doses 100 or 1,000-fold higher than a Tolerable Daily Intake for humans had no effect on its testicular development.     

NOTCH signaling in Sertoli cells regulates gonocyte fate

Gonocytes (or prospermatogonia) are the precursors to spermatogonial stem cells (SSCs), which provide the foundation for spermatogenesis through their ability to both self-renew and generate daughter cells. Despite their relative importance, the regulatory mechanisms that govern gonocyte maintenance and transition to SSCs are poorly understood. Recently, we reported that constitutive activation of NOTCH1 signaling in Sertoli cells causes gonocyte exit from quiescence—the first suggestion of the potential role of this signaling pathway in the testis.

This Extra View will review what is known about NOTCH signaling, particularly in Sertoli cells and germ cells in the testes, by providing a background on germ cell biology and a summary of our recently published data on NOTCH1 signaling in Sertoli cells. We also describe additional data showing that aberrant proliferation and differentiation of gonocytes in response to constitutive activation of NOTCH1 signaling in Sertoli cells involves de novo expression of cell cycle proteins and a marked upregulation of the KIT receptor. These data further suggest that NOTCH signaling orchestrates a dynamic balance between maintenance and differentiation of gonocytes in the perinatal testis.     

Sertoli cell behaviors in developing testis cords and postnatal seminiferous tubules of the mouse

Sertoli cells are the primary structural component of the fetal testis cords and postnatal seminiferous tubules. Live imaging technologies facilitate the visualization of cell morphologies and behaviors through developmental processes. A transgenic mouse line was generated using a fragment of the rat Gata4 gene to direct the expression of a dual-color fluorescent protein reporter in fetal and adult Sertoli cells. The reporter encoded a red fluorescent protein, monomeric Cherry (mCherry), fused to histone 2B and enhanced green fluorescent protein (EGFP) fused to a glycosylphosphatidylinositol sequence, with a self-cleaving 2A polypeptide separating the two fusion proteins. After translation, the red and green fluorescent proteins translocated to the nucleus and plasma membrane, respectively, of Sertoli cells. Transgene expression in testes was first detected by fluorescent microscopy around Embryonic Day 12.0. Sertoli cell division and migration were visualized during testis cord formation in organ culture. Initially, the Sertoli cells had mesenchyme-like morphologies and behaviors, but later, the cells migrated to the periphery of the testis cords to become epithelialized. In postnatal seminiferous tubules, Sertoli nuclei were evenly spaced when viewed from the external surface of tubules, and Sertoli cytoplasm and membranes were associated with germ cells basally in a rosette pattern. This mouse line was bred to previously described transgenic mouse lines expressing EGFP in Sertoli cytoplasm or a nuclear cyan fluorescent protein (Cerulean) and mCherry in plasma membranes of germ cells. This revealed the physical relationship between Sertoli and germ cells in developing testis cords and provided a novel perspective on Sertoli cell development.     

mRNAs encoding a von Ebner's-like protein and the Huntington disease protein are induced in rat male germ cells by Sertoli cells

The success of spermatogenesis is dependent upon closely coordinated interactions between Sertoli cells and germ cells. To identify specific molecules that mediate interactions between somatic cells and germ cells in the rat testis, Sertoli cell-germ cell co-cultures and mRNA differential display were used. Two cDNAs, clone 1 (660 nucleotides) and clone 2 (390 nucleotides) were up-regulated when Sertoli cells were co-cultured with pachytene spermatocytes or round spermatids. Northern blot analyses confirmed the differential display expression patterns. Sequence analyses indicated that clone 1 was similar to a von Ebner's gland protein (87% at the nucleotide level and 80% at the amino acid level) and clone 2 was identical to a region of the Huntington disease protein. The von Ebner's-like protein mRNA was induced after 4 h of co-culture, while the Huntington disease protein required 18 h of co-culture for expression. The von Ebner's-like protein was induced in germ cells by a secreted Sertoli cell factor(s) smaller than 10 kDa that is sensitive to freezing and thawing or boiling. The Huntington disease protein was induced in germ cells by a Sertoli cell secreted factor(s) larger than 10 kDa which survives freezing and thawing, but is inactivated by boiling.     

Densin is a novel cell membrane protein of Sertoli cells in the testis

Cell-cell interactions between Sertoli cells and germ cells are crucial for the maturation of germ cells in spermatogenesis but the structural and functional aspects of the interactions remain to be fully elucidated. Densin is a junction protein suggested to play a role in establishment of specific cell-cell contacts in the post-synaptic densities of the brain and the slit diaphragm of the kidney podocyte. In the present study, densin was discovered to be expressed in the testis of the man and the mouse. Expression of densin at the gene and the protein level was studied by using RT-PCR and Western blotting analyses, and the localization of densin was explored with immunofluorescence staining. RT-PCR and Western blotting analyses showed that densin is expressed at the gene and the protein levels. Immunofluorescence staining localized the expression of densin to the cell membranes of Sertoli cells suggesting that densin may be an adherens junction protein between Sertoli cells and developing germ cells. Densin is a novel testicular protein expressed in the cell membranes of Sertoli cells. Its functional role remains to be assessed.     

Multiple effects of retinoids on the development of Sertoli, germ, and Leydig cells of fetal and neonatal rat testis in culture

We investigated the effect of retinoids on the development of Sertoli, germ, and Leydig cells using 3-day culture of testes from fetuses 14.5 and 18.5 days post-conception (dpc) and from neonates 3 days postpartum (dpp). Addition of 10(-6) M and 3.10(-8) M retinoic acid (RA) caused a dose-dependent disruption of the seminiferous cords in 14.5-day-old fetal testes, without any change in the 5-bromo-2'-deoxyuridine (BrdU) labeling index of the Sertoli cells. RA caused no disorganization of older testes, but it did cause hyperplasia of the Sertoli cells in 3-dpp testes. Fragmentation of the Sertoli cell DNA was not detected in control or RA-treated testes at any age studied. The cAMP produced in response to FSH was significantly decreased in RA-treated testes for all studied ages. Both 10(-6) M and 3.10(-8) M RA dramatically reduced the number of gonocytes per 14.5-dpc testis. This resulted from a high increase in apoptosis, which greatly exceeded the slight increase of mitosis. RA caused no change in the number of gonocytes in testes explanted on 18.5 dpc (the quiescent period), whereas it increased this number in testes explanted on 3 dpp (i.e., when gonocyte mitosis and apoptosis resume). Lastly, RA and retinol (RE) reduced both basal and acute LH-stimulated testosterone secretion by 14.5-dpc testis explants, without change in the number of 3beta-hydroxysteroid dehydrogenase-positive cells per testis. Retinoids had no effect on basal or LH-stimulated testosterone production by older testes. In conclusion, RE and RA are potential regulators of the development of the testis and act mainly negatively during fetal life and positively during the neonatal period on the parameters we have studied.     

Effect of an acute exposure of rat testes to gamma rays on germ cells and on Sertoli and Leydig cell functions.

Germ cells and Sertoli and Leydig cell functions were studied from 7 to 180 days after an acute exposure of 2-month-old rat testes to 9 Gy of gamma rays. Body weight, testis and epididymal weights were recorded. Sertoli cell parameters (androgen-binding protein, ABP, in caput epididymis and plasma follicle stimulating hormone, FSH) and Leydig cell parameters (plasma luteinizing hormone, LH, testosterone and prostate and seminal vesicle weights) were determined together with the number of germ cells and Sertoli cells. Irradiation did not affect body weight but significantly reduced testicular and epididymal weights from day 7 and day 15 post-irradiation respectively. The cells killed by irradiation were mainly spermatogonia and preleptotene spermatocytes engaged in replicating their DNA at the time of exposure, but all spermatocytes seemed damaged as they gave abnormal descendent cells. By day 34, only elongated spermatids remained in a few tubules and thereafter very little regeneration of the seminiferous epithelium occurred, except for one rat which showed a better regeneration. Levels of ABP decreased by day 15 when the germ cell depletion had reached the pachytene spermatocytes, whereas FSH and LH levels rose when the number of elongated spermatids decreased. Levels of testosterone and the weight of the seminal vesicles did not change; occasionally, the prostate weight was slightly reduced. These results support our hypothesis that pachytene spermatocytes and elongated spermatids are involved in influencing some aspects of Sertoli cell function in the adult rat.     

Survival and proliferation of rat gonocytes in vitro

Quiescent gonocytes were isolated from fetal testes of rat 18-day post coitum and cultured alone or on monolayers of somatic cells from different origins. The gonocytes specifically adhered to Sertoli cells, isolated from 21 to 23-day-old rat testes; this adherence was necessary for their survival in vitro. Addition of follicle-stimulating hormone and testosterone to these cultures did not increase the viability of the gonocytes. Serum was found to be deleterious to the germ cells. Electron-microscopic examination of Sertoli-cell-gonocyte co-cultures revealed the presence of numerous adhesion plaques between these cells, indicating that Sertoli cells and gonocytes are able to communicate in vitro. Gonocytes, in co-culture with Sertoli cells, were viable for at least 9 days. The gonocytes did not spontaneously resume proliferation. The simple culture system described in the present paper should be useful in studying the nature of the factors that are responsible for sending the quiescent gonocytes into the cell cylce and for stimulating the formation of A spermatogonia, a process characterizing the start of spermatogenesis.     

Development of Fetal Testicular Cells in Androgen Receptor Deficient Mice

During mammalian development, androgens produced by the fetal testis are the most important hormones controlling the masculinization of the reproductive tract and the genitalia. New findings show that the male germ line is the most sensitive to anti-androgenic endocrine disruptors during the embryonic period. In a recent study, we reported that endogenous androgens physiologically control germ cell growth in the male mouse fetus during early fetal life. In the present study, we extended this result by showing the presence of a functional androgen receptor in the gonocytes in the latter part of the fetal life. We also studied the effect of androgens on the development of the somatic testicular cells using the Tfm mice which carry a naturally inactivating mutation of the androgen receptor. Fetal Leydig cell are largely independent of endogenous androgens during fetal development whereas fetal Sertoli cell number is decreased following a default of peritubular myoid cells differenciation. They also point to the gonocyte as a special target for androgens during the embryonic period and indicate a novel mechanism of androgen action on gonocytes. Elucidation of this new pathway in the fetal testis will clarify not only fetal testis physiology but also the effects of environmental anti-androgens that act during fetal life and open new perspectives for future investigations into the sensitivity of fetal germ cell to androgens.     

Ultrastructure of germ cell development in the human fetal testis

Summary Electron-microscopic examination of the human fetal testis between 10 and 20 weeks gestation reveals the presence of two distinct cell types within the tubules: Sertoli cells and germ cells. The latter are distinguished by their spherical shape, smooth nuclear membranes, globular mitochondria and paucity of cytoplasmic organelles. The gonocytes, or primitive germ cells, occur as single cells in the central portions of the tubules. Their chromatin is finely granular and evenly dispersed. Nucleoli are centrally placed and of uniform electron density. Various stages in the migration of gonocytes to the tubular periphery are indicated by the extension of cytoplasmic processes toward the basal lamina. Bands of microtubules are present within the processes. Spermatogonia are arranged in pairs and groups at the tubular periphery. They lack the nucleolar and mitochondrial characteristics of adult spermatogonia. Except for slight changes in chromatin density and nucleolar structure, the fetal spermatogonia retain the ultrastructural characteristics of gonocytes. Intercellular bridges connect adjacent spermatogonia. Degeneration affecting large numbers of germ cells, but primarily gonocytes, begins with nuclear infolding and chromatin condensation and eventually involves both nuclear and cytoplasmic structures. The degenerated cells are removed by phagocytosis by adjacent Sertoli cells. Large phagosomes are present in the cytoplasm of many of the Sertoli cells.Supported by a grant from the Ford Foundation and by General Research Support Grant RR055511 from the National Institutes of Health. Technical assistance was provided by Mrs. Lucy A. Conner.     

Germ cell differentiation and proliferation in the developing testis of the South American plains viscacha, Lagostomus maximus (Mammalia, Rodentia)

Cell proliferation and cell death are essential processes in the physiology of the developing testis that strongly influence the normal adult spermatogenesis. We analysed in this study the morphometry, the expression of the proliferation cell nuclear antigen (PCNA), cell pluripotency marker OCT-4, germ cell marker VASA and apoptosis in the developing testes of Lagostomus maximus, a rodent in which female germ line develops through abolished apoptosis and unrestricted proliferation. Morphometry revealed an increment in the size of the seminiferous cords with increasing developmental age, arising from a significant increase of PCNA-positive germ cells and a stable proportion of PCNA-positive Sertoli cells. VASA showed a widespread cytoplasmic distribution in a great proportion of proliferating gonocytes that increased significantly at late development. In the somatic compartment, Leydig cells increased at mid-development, whereas peritubular cells showed a stable rate of proliferation. In contrast to other mammals, OCT-4 positive gonocytes increased throughout development reaching 90% of germ cells in late-developing testis, associated with a conspicuous increase in circulating FSH from mid- to late-gestation. TUNEL analysis was remarkable negative, and only a few positive cells were detected in the somatic compartment. These results show that the South American plains viscacha displays a distinctive pattern of testis development characterized by a sustained proliferation of germ cells throughout development, with no signs of apoptosis cell demise, in a peculiar endocrine in utero ambiance that seems to promote the increase of spermatogonial number as a primary direct effect of FSH.     

Sertoli cell development and function in an animal model of testicular dysgenesis syndrome

Pregnancy exposure to di(n-butyl) phthalate (DBP) in rats induces a testicular dysgenesislike syndrome (TDS) in male offspring. Earlier studies suggested altered Sertoli cell development/maturation may result, especially in testes that become cryptorchid. This study quantitatively assessed Sertoli cell numerical and functional development in DBP-exposed rats and compared (unilaterally) cryptorchid and scrotal testes. Pregnant rats were gavaged with 500 mg/kg/day DBP or corn oil from embryonic (E) Days 13.5 to 21.5. Male offspring were sampled on E21.5 or Postnatal Day 6, 10, 15, 25, or 90. Sertoli cell number in DBP-exposed males was reduced by approximately 50% at E21.5 but recovered to normal by Days 25-90, accompanied by significant changes in plasma inhibin B and testosterone levels. Sertoli cell maturational development in DBP-exposed males, assessed using five protein markers (anti-müllerian hormone, cytokeratin, androgen receptor, CDKN1B, and Nestin), was largely normal, with some evidence of delayed maturation. However, in adulthood, Sertoli cells (SC) in areas lacking germ cells (Sertoli cell-only [SCO] tubules) often exhibited immature features, especially in cryptorchid testes. Sertoli cells in DBP-exposed animals supported fewer germ cells during puberty, but this normalized in scrotal testes by adulthood. Scrotal and especially cryptorchid testes from DBP-exposed animals exhibited abnormalities (SCO tubules, focal dysgenetic areas) at all postnatal ages. Cryptorchid testes from DBP-exposed animals exhibited more Sertoli cell abnormalities at Day 25 compared with scrotal testes, perhaps indicating more severe underlying Sertoli cell malfunction in these testes. Our findings support the concept of altered Sertoli cell development in TDS, especially in cryptorchid testes, but show that maturational defects in Sertoli cells in adulthood most commonly reflect secondary dedifferentiation in absence of germ cells.     

Sertoli cell vacuolization and abnormal germ cell adhesion in mice deficient in an inositol polyphosphate 5-phosphatase

The dynamic nature of cellular interactions during differentiation of germ cells and their translocation from the basement membrane to the lumen of the seminiferous tubules requires the existence of complex and well-regulated cellular adhesion mechanisms in the testis. Successful migration of the developing germ cells is characterized by dynamic breakage and reformation of cadherin-containing adherens junctions between the germ cells and Sertoli cells, the polarized somatic cells of the testis that support and nourish the developing gametes. Here, we demonstrate the accumulation of abnormally swollen, actin-coated, endosome-like structures that contain intact adherens junctions and stain positive for N-cadherin and beta-catenin in the Sertoli cell cytosol of mice deficient in Inpp5b, an inositol polyphosphate 5-phosphatase. Simultaneous to the formation of these abnormal structures, developing germ cells are prematurely released from the seminiferous epithelium and sloughed into the epididymis. Our results demonstrate a role for Inpp5b in the regulation of cell adhesion in the testis and in the formation of junctional complexes with neighboring cells, and they emphasize the important and essential role of phosphoinositides in spermatogenesis.