Expression of a novel factor, short-type PB-cadherin, in Sertoli cells and spermatogenic stem cells of the neonatal rat testis.

In the rodent testis, contact-mediated interactions between gonocytes, or neonatal stem cells, and Sertoli cells are critical for development. Previously, we showed that the neural cell adhesion molecule (NCAM) serves as a Sertoli cell-gonocyte attachment factor in neonates. Its expression decreases dramatically by 1 week of age and eventually disappears in vivo, and appears to be down-regulated by thyroid hormone (tri-iodothyronine (T(3))). In this study, we used a cDNA microarray to screen for additional adhesion factors which might be important in testes of developing rats and detected expression of a novel factor, short-type PB-cadherin (STPB-C). Next, RT-PCR was used to generate cDNA for STPB-C from total RNA isolated from co-cultures, cDNA was cloned into pPCR-Script Amp SK(+) cloning vector, and plasmid DNA was isolated and sequenced to confirm the fidelity of the STPB-C cDNA portion of the plasmid. In situ hybridization analyses of testicular sections indicated that STPB-C expression in neonates is localized in the cytoplasm of many, but not all, gonocytes and in the cytoplasm of most of the surrounding Sertoli cells. Parallel hybridizations carried out on co-cultures also demonstrated a strong cytoplasmic signal in some gonocytes and in the great majority of the Sertoli cells of the underlying monolayer. With Northern analyses we found that STPB-C is expressed in vivo at high levels between days 1 and 5, with a subsequent large drop by day 10 and thereafter, suggesting that its expression may be associated with Sertoli or germ cell differentiation. Subsequent analyses of co-cultures exposed under a variety of conditions to T(3) suggest that, unlike NCAM, STPB-C is not regulated by this hormone. Next, we studied production of STPB-C protein by using an antiserum recognizing a peptide sequence unique to this factor in Western blotting and in immunolocalization. Signal was detected both intracellularly and at cell surfaces in most Sertoli cells and many gonocytes, although many of the latter cell type were also found to be negative for the protein, suggesting a potential role for STPB-C in survival and further development of some of these germ cells from which all subsequent spermatogenic cells originate.

Gonocyte-Sertoli cell interactions during development of the neonatal rodent testis.

During neonatal testicular development in the rat, events critical for subsequent germ cell development occur that set the stage for fertility later in life. Some gonocytes resume mitotic activity and/or migrate to the surrounding basal lamina, and use of a carefully defined Sertoli cell-gonocyte coculture system indicates that these crucial events occur without added factors or hormones and are hence likely to depend on interaction with adjacent Sertoli cells. Coupling of the Kit receptor protein on gonocytes to stem cell factor from Sertoli cells is vital for successful migration by gonocytes, as antagonism of the former suppresses and addition of the latter stimulates gonocyte migration. During the neonatal period, intercellular adhesion is modified in a developmental manner such that neural cell adhesion molecule (NCAM) is the main adhesive molecule expressed and functioning at birth, with a progressive decline as development proceeds. This decline in NCAM expression is supported by the addition of exogenous 3,3',5-triiodothyronine in vitro, and because this factor is recognized as supporting Sertoli cell differentiation, it seems likely that changing intercellular adhesion is a function of progressive development of Sertoli cells. Other avenues whereby maturing testicular cells influence each other doubtless exist, including secretion of growth factors and other peptides and developmentally important changes in the makeup of the extracellular matrix, which Sertoli cells and gonocytes contact. Continued investigation in these areas will be very valuable in enlarging our understanding of how neonatal testicular development provides the basis for successful spermatogenesis.

A single dose of Di-(2-ethylhexyl) phthalate in neonatal rats alters gonocytes, reduces sertoli cell proliferation, and decreases cyclin D2 expression.

In this study, we explored the impact on both Sertoli cells and gonocytes of a single, relatively low dose of di-(2-ethylhexyl) phthalate (DEHP; 20-500 mg/kg) administered in vivo to 3-day-old rat pups. In parallel, we assessed the potential for two immediate metabolites of DEHP to produce similar testicular changes and began to explore the possible mechanisms involved. Morphological examination revealed the presence of many abnormally large, multi-nucleated germ cells by 24 h posttreatment with DEHP and with its metabolite, mono-ethylhexyl phthalate (MEHP), but not with another metabolite, 2-ethylhexanol (2-EH; all at 1.28 mmol/kg) or with vehicle alone. These cells persisted through 48 h posttreatment, the longest time point examined in our study. We also assessed the rate of Sertoli cell proliferation in pups at intervals after dosage with either chemical or vehicle by administering bromodeoxy uridine (BrdU) 3 h before euthanasia. By 24 h after treatment with DEHP or MEHP, but not 2-EH or vehicle, the number of BrdU-labeled Sertoli cells was obviously diminished in testicular sections. Quantitation of DEHP-treated pups and controls indicates that a dose-response relationship exists between chemical treatment and labeling index (LI) of Sertoli cells, with a LI at the highest DEHP dose tested that was only 20% of that in controls. In addition, when we examined the time course of the effect of an intermediate dose of DEHP, we found that there the LI of Sertoli cells rebounds by 48 h after dosage, when we found the rate of proliferation in treated pups to be significantly higher than in controls. We also explored the potential mechanism involved in the response to DEHP and found serum levels of FSH to be unaffected by the chemical. In addition, study of cell cycle-related proteins including p27kip1 and cyclins D1, D2, and D3 with Western and Northern analysis indicated that cyclin D2 mRNA is specifically down-regulated by DEHP in a dose-dependent manner, and this decrease is manifest as a small, transient but reproducible reduction in the amount of cyclin D2 protein detectable in samples from treated pups compared to controls. Our findings characterize the changes in neonatal Sertoli cells and gonocytes that follow in vivo to low levels of DEHP and its metabolite, MEHP, as well as providing new information on the underlying mechanism and highlighting the extreme sensitivity of the neonatal testis to injury by this toxicant.

Thyroid hormone down-regulates neural cell adhesion molecule expression and affects attachment of gonocytes in Sertoli cell-gonocyte cocultures.

Contact-mediated interactions between Sertoli cells and gonocytes are important for testicular development. Specifically, down-regulation of neural cell adhesion molecule (NCAM)-based intercellular adhesion during postnatal maturation is likely to be important for appropriate differentiation of testicular cells. Besides NCAM, P-cadherin is also present in neonatal testicular cords, at least in mice, and seems to disappear from the seminiferous epithelium after the first postnatal week. Another factor known to be important in regulating development of the neonatal testis is thyroid hormone (T3). T3 is involved in control of Sertoli cell proliferation and differentiation. Therefore, we examined the effect(s) of T3 on adhesive factors found within the testis using Sertoli cells and gonocytes isolated from neonates and maintained in coculture. T3 (100 nM) down-regulated NCAM expression in vitro, as assessed by Western blotting and immunofluorescent staining. This contrasted with the continued expression of NCAM in cultures without added T3 but mimicked the disappearance of NCAM from the neonatal rat testis in vivo. In addition, Western analysis confirmed that P-cadherin is highly expressed in the developing rat testes, as it is in those of mice. We found that P-cadherin is strongly expressed in gonocytes and weakly expressed in Sertoli cells. Moreover, unlike NCAM, P-cadherin expression diminishes with time in vitro in the absence of added hormones. In parallel with our observations for NCAM, expression of P-cadherin was also apparently decreased by T3 (100 nM). Subsequent quantitative analyses of cultures exposed to a range of T3 levels (0.1-100 nM) indicated that T3 causes detachment of many gonocytes in a dose- and time-dependent manner (approximately 80% detached at 100 nM). In addition, Western blotting indicated that lower concentrations of T3 down-regulate NCAM but not P-cadherin. From this we conclude that the apparent decrease in P-cadherin induced by 100 nM T3 and detected on Western blots reflects loss of gonocytes. In contrast, even low levels of T3 appear to down-regulate NCAM production before any significant detachment of gonocytes. Finally, low levels of T3 that did not affect numbers of adherent Sertoli cells nevertheless caused detachment of gonocytes. Thus, our observations identify T3 as a regulator of NCAM expression in neonatal testicular cells and as a modifier of gonocyte/Sertoli cell adhesion in vitro.

Expression of 140-kDa neural cell adhesion molecule in developing testes in vivo and in long-term Sertoli cell-gonocyte cocultures.

The basis for cell-cell adhesion in the seminiferous epithelium of the developing testis is doubtless critical in supporting events that are essential for the onset and maintenance of normal spermatogenesis. In this study, we applied immunoblotting and immunolocalization approaches for the following reasons: 1) to ask whether neural cell adhesion molecule (NCAM) underlies cell-cell interactions in vivo, as we previously showed for cells in vitro, 2) to characterize the isoform or isoforms of NCAM expressed during testicular development, and 3) to study NCAM expression in long-term Sertoli cell-gonocyte cocultures and to compare and contrast this pattern of expression with that in vivo. Our findings indicate that NCAM is found ubiquitously at cell-cell interfaces within the seminiferous cord from birth through day 10 and thereafter is restricted to interstitial cells. Moreover, only polysialic acid-negative 140-kDa NCAM is expressed in the testis or in coculture, an isoform whose properties are compatible with the concept of NCAM as both a direct modifier of cell function and an indirect influence on cell responses mediated by other external factors. In addition, we found that germ cells, potentially gonocytes or Type A spermatogonia, persist in long-term cocultures maintained for 15 days after isolation from 5-day-old rat pups and that NCAM continues to be expressed at high levels in these cultures. This observation is in marked contrast to our observation that NCAM gradually decreases and eventually disappears in vivo by postnatal day 15. Thus, our findings indicate that 140-kDa NCAM is prominent in neonatal testes but is down-regulated by as yet unidentified mechanisms thereafter. Our findings also indicate that down-regulation of NCAM fails to occur in hormone- and serum-free Sertoli cell-germ cell cocultures.

Effects of relatively low levels of mono-(2-ethylhexyl) phthalate on cocultured Sertoli cells and gonocytes from neonatal rats.

Di-(2-ethylhexyl) phthalate (DEHP), one of the abundant man-made environmental chemicals, induces testicular damage in both developing and adult animals. However, the nature and mechanism underlying the action of phthalates on testicular development remain largely unexplored. In the present study, we used cocultures of neonatal Sertoli cells and gonocytes (precursors of spermatogonia) to characterize in detail the effects of mono-(2-ethylhexyl) phthalate (MEHP; the active metabolite of DEHP) on these cells and to explore the underlying mechanism(s). Sertoli cells and gonocytes were isolated from rat pups on the 2nd day after birth, cocultured, and exposed to MEHP at concentrations of 0.01, 0.1, or 1.0 microM, or to 0.5% DMSO (vehicle control), or 10 microM DEHP (negative control) for a total of 48 h. We found that exposure to MEHP induced gonocyte detachment from the Sertoli cell monolayers in a time- and dose-dependent manner. When exposed to 1.0 microM MEHP, many gonocytes started to detach after 12 h of exposure and most gonocytes were lost during the media change at 24 h. Gonocyte detachment was also observed in cocultures treated with 0.1 microM MEHP for 24 h of exposure, but not in cultures treated with 0.01 microM MEHP for 48 h. Detached gonocytes were viable as indicated by their ability to exclude trypan blue. Furthermore, when proliferation of cultured Sertoli cells was detected by BrdU labeling and subsequently quantified, we found that exposure to 0.1 or 1.0 microM MEHP for 48 h resulted in a decrease in labeling indices of 33.6 and 83.6%, respectively, compared to the vehicle control (p < 0.01), while the labeling index was unchanged by treatment with 0.01 microM MEHP. In addition, we also tested the potential effect of MEHP on FSH-stimulated Sertoli cell proliferation by simultaneously treating cultures with 200 ng/ml human FSH and different concentrations of MEHP for 48 h. Exposure to 0.1 or 1.0 microM MEHP resulted in decreases of 24.2 and 74.2%, respectively, in FSH-stimulated Sertoli cell proliferation (p < 0. 01). Furthermore, MEHP also inhibited dibutyl cAMP-stimulated Sertoli cell proliferation, regardless of whether dibutyl cAMP was added to the cultures before or at the same time as MEHP. Finally, addition of FSH or dibutyl cAMP had no effect on MEHP-induced gonocyte detachment, and none of the observed effects on either Sertoli cells or gonocytes were detected in control cultures treated with 0.5% DMSO only or with 10 microM DEHP. Therefore, short exposure to low levels of MEHP disrupted adhesion of gonocytes to Sertoli cells and inhibited both basal and FSH-stimulated Sertoli cell proliferation in a dose-dependent manner. The lowest effective dose of MEHP in vitro was 0.1 microM, which is about 10- to 1, 000-fold lower than the dose shown to affect Sertoli cells from prepubertal animals. Moreover, our data indicate that MEHP impairs division of neonatal Sertoli cells by acting at a post-cAMP site in the FSH-response pathway or via a mechanism independent of FSH. These data provide direct new evidence that relatively low levels of MEHP disrupt Sertoli cell-gonocyte physical interactions and suppress Sertoli cell proliferation in neonates via mechanisms specific to neonatal testis where the foundations of adult fertility are established. The results also highlight the neonatal period of testicular development as one particularly sensitive to environmental chemicals.

Use of in vitro systems to study male germ cell development in neonatal rats.

The aim of this review is to summarize ways in which in vitro approaches have allowed us to investigate several aspects of gametogenesis in the male. In our laboratory, we have established both organ culture and cell co-culture methodologies and applied them to questions focused on cellular and molecular events important for development of primitive spermatogonia, or gonocytes, in testes of neonatal rats. We have described their postnatal reinitiation of mitosis and their migration to the basal lamina in anticipation of basal compartment formation and, through use of these in vitro systems, we have identified several mechanisms regulating these processes. These include matrix influence on mitosis and migration, adhesive mechanisms active between gonocytes and Sertoli cells, and involvement of the Kit receptor on germ cells and its ligand from Sertoli cells in supporting gonocyte migration, as described below.

Expression of the c-kit gene is critical for migration of neonatal rat gonocytes in vitro.

Rat gonocytes migrate to the basement membrane during the first postnatal week, a change in position crucial for their survival. These cells express the c-kit gene from the day of birth through Day 5 in vivo and develop the ability to migrate in Sertoli cell-gonocyte cocultures. In this study, we asked whether c-kit expression and synthesis of Kit protein are required for pseudopod production by gonocytes in vitro. To determine whether gonocyte migration in vitro is invariably accompanied by c-kit expression, we quantified percentages of gonocytes expressing c-kit with increasing time in vitro and correlated these data with pseudopod development by individual cells. We also determined the effect of exposure to Kit antibodies on gonocyte migration in vitro, and, conversely, asked whether addition of exogenous stem cell factor (SCF), the Kit ligand, stimulates pseudopod development. We found that 1) increasing numbers of gonocytes express c-kit with increasing time in vitro; 2) once these cells begin migrating in vitro, the appearance of a pseudopod on a gonocyte is absolutely correlated with kit expression by that cell; 3) incubating cocultures with Kit antibodies significantly reduces the number of cells with pseudopods, without any detectable decrease in numbers of gonocytes; and 4) addition of exogenous SCF to cocultures prepared on Day 5 results in a transient but significant increase in the percentage of gonocytes with pseudopods even though we found that Sertoli cells in the cultures produce endogenous SCF. Thus, our findings provide evidence to support a role for c-kit expression by neonatal gonocytes and, presumably, SCF expression by neonatal Sertoli cells in stimulating migration of these germ cells in vitro.

Hst7: a male sterility mutation perturbing sperm motility, flagellar assembly, and mitochondrial sheath differentiation.

Hst7, a mouse hybrid sterility locus, has been mapped in close linkage to four other hybrid sterility loci, on proximal chromosome 17 within the t complex. When an allele (s) of Hst7 from the species Mus spretus is crossed into the Mus musculus domesticus (laboratory mouse) background, all male offspring are sterile. This occurs regardless of whether the Hst7 allele on the other chromosome 17 homolog is wild-type (+) or an allele (t) derived from the structurally variant homolog known as a t haplotype. Males of the Hst7 genotype s/+ produce sperm that, after release from the cauda epididymis, display moderate asthenospermia (straight line velocity = 49 +/- 4 microm/second, significantly lower than 102 +/- 7 microm/second for congenic wild-type controls) and normal morphology. However, males of the Hst7 genotype s/t produce sperm whose forward movement is below the detectable limit of the sperm motion analysis system. In addition, these sperm exhibit a variety of flagellar abnormalities, with about one third having normal heads attached to sacklike caudal regions. These sacks consist of membrane-delimited cytoplasm containing disorganized and/or misshapen axonemal elements. The remainder of the sperm from s/t mice have flagella with seemingly normal axonemes, although many exhibit enlarged areas of cytoplasm in their midpieces with extra layers of misaligned mitochondria. The s/t sperm mitochondria also display diffuse and vacuolated matrices reminiscent of meiotic germ cell and spermatid mitochondria. Observations of developing spermatids in the s/t testis reveal an unusual phenotype in which gaps of varying length occur in the mitochondrial wrapping of the midpiece. These data suggest that both the s and t alleles of Hst7 are defective alleles that contribute differentially to the severe asthenospermia phenotype and interact genetically to perturb flagellar development.

Gonocytes in testes of neonatal rats express the c-kit gene.

Information gathered from mutant mouse models and from studies on normal puberal and adult animals points to the product of the c-kit gene, a tyrosine kinase surface receptor, and the kit-ligand (KL) as important for gametogenesis in males. In fetuses, KL serves as a survival factor for primordial germ cells, at least in vitro, and in adults activity of the c-kit gene has been indirectly related to survival and subsequent development of differentiating spermatogonia. However, because of the structural complexity of the seminiferous epithelium in adults, c-kit mRNA has not yet been definitively localized to one or more types of spermatogenic cells. In addition, no information is currently available regarding the possible involvement of the c-kit protein and its ligand in mediating germ cell development and/or Sertoli-germ cell interactions immediately after birth when events critical for later onset of spermatogenesis are ongoing. Thus, the aims of the current study were (1) to determine whether the c-kit gene is expressed in testes of neonatal and adult rats and, if so, by what specific cell types, and (2) to determine if those cells expressing the gene also produce the c-kit receptor protein. For this, we isolated total RNA from testes of pups aged days 1-5 and from adult rat testes, and probed for the presence of c-kit mRNA with Northern analysis. We identified the cells containing the c-kit message by carrying out in situ hybridization with digoxigenin-labeled probes, thus allowing the colorimetric signal to be assigned beyond doubt to individual cells in sections of testes. We also utilized Western analysis and immunolocalization to confirm the presence of the c-kit receptor protein in testes at these ages and to identify those cells types producing it. Our findings indicate that (1) neonatal gonocytes express the c-kit gene and produce the receptor protein on postnatal days 1 through 5, spanning the time when they resume dividing and migrating, and (2) spermatogonia and, to a lesser extent, spermatocytes and spermatids of adults express the gene but c-kit protein is present in detectable amounts only in spermatogonia and possibly a few early primary spermatocytes.

Temporal patterns of A-myb and B-myb gene expression during testis development.

We recently reported the cloning and sequencing of the mouse A-myb proto-oncogene cDNA and the abundant expression of this mRNA primarily in the testis of adult mice. The A-myb mRNA is detectable by in situ hybridization specifically in the spermatogenic cells, and is downregulated during terminal differentiation. A low level of expression is observed in a few other tissues, including ovary, spleen and brain. We have extended those studies by examining A-myb and B-myb expression during testis development in the mouse. The A-myb and B-myb genes are both expressed in a cell- and stage-specific manner during testis development. The B-myb mRNA is expressed most highly in gonocytes of the fetal testis and in spermatogonia and early spermatocytes in the adult. B-myb expression decreases at day 18 post partum, coincident with the initial appearance of late pachytene spermatocytes. B-myb expression was also detectable in some interstitial cells of the fetal and adult testis. The A-myb mRNA was not detectable by in situ hybridization in fetal day 15.5 gonocytes but was detectable at a low abundance by RT-PCR in fetal and newborn mice. A-myb mRNA expression increased at post-natal day 10, when primary spermatocytes first appear. In the adult, the A-myb mRNA was expressed highly in a sub-population of spermatogonia and in primary spermatocytes, but was not detectable in spermatids. This expression of A-myb is consistent with the meiotic arrest that is observed in A-myb-deficient male mice. We conclude that B-myb may play a critical role in controlling the proliferation or differentiation of gonocytes and spermatogonia and possibly the somatic lineages as well, whereas A-myb is required for progression through the first meiotic prophase. These distinct roles for B-myb and A-myb during spermatogenesis may reflect distinct transactivation potentials of the two proteins. Further studies to determine the functions of A-myb and B-myb in the developing testis should improve our understanding of the molecular events associated with spermatogenesis and differentiation of the Sertoli and other somatic cell types of the testis.

Sperm from mice carrying two t haplotypes do not possess a tyrosine phosphorylated form of hexokinase.

Mouse sperm contain a tyrosine phosphorylated form of hexokinase type 1 (HK1; Kalab et al., 1994: J Biol Chem 269:3810-3817) that has properties consistent with an integral plasma membrane protein. Furthermore, this tyrosine phosphorylated form of HK1 has an extracellular domain and HK1 is localized to both the head and flagellum of nonpermeabilized cells (Visconti et al., 1995c). We have characterized HK1 in mature sperm from sterile tw32/tw5 mice (mutant sperm) that have defects in motility and sperm-egg interaction (Johnson et al., 1995: Dev Biol 168:138-149). Immunoprecipitation of mouse sperm extracts with an antiserum made against purified rat brain HK1 demonstrates the presence of HK1 in mutant sperm. Various biochemical and immunofluorescence assays indicate that at least a portion of the HK1 present in these cells is an integral membrane protein with an extracellular domain located on the sperm head and flagellum. However, immunoblot analysis with anti-phoshotyrosine antibodies demonstrates that HK1 in mutant sperm is not tyrosine phosphorylated. Northern blot and RT-PCR analysis does not indicate any obvious abnormalities in the transcription of somatic or germ cell-specific HK1 isoforms in mutant testes, and RFLP analysis of recombinant mice indicates that no genes specifying HK1 isoforms are located on chromosome 17. We have mapped the locus responsible for the lack of tyrosine phosphorylation of HK1 mutant sperm to the most proximal (to the centromere) of the four inversions within the t haplotype. A male sterility factor is located in this same inversion (Lyon, 1986: Cell 44:357-363). Since the mutant sperm are unable to complete fertilization, there could be a relationship between sterility and the lack of tyrosine phosphorylation of HK1 in these mutant sperm.

NCAM mediates adhesion between gonocytes and Sertoli cells in cocultures from testes of neonatal rats.

During neonatal development of the rat testis, gonocytes resume mitosis and display renewed motility to migrate toward the basal lamina, two events that occur in vitro when these cells are cocultured with Sertoli cells. However, although substantial evidence suggests that development of gonocytes depends on Sertoli cells, little is known of how these cell types interact beyond our previous observations that they communicate via gap junctions and adhere avidly to each other. In the present study, we utilized several approaches to examine the mechanism by which gonocytes adhere to Sertoli cells in vitro. First, we characterized this attachment in general by (1) determining its susceptibility to brief trypsinization in decreasing concentrations of Ca2+, (2) assessing the ability of gonocytes to adhere to Sertoli cells at reduced temperature, and (3) examining the effect of phospholipase C treatment on the number of gonocytes attached to a Sertoli cell monolayer. Because the findings suggested that a non-cadherin mechanism is involved, we used immunofluorescence to identify the presence of neural cell adhesion molecule (NCAM) at virtually all gonocyte-Sertoli cell (and Sertoli cell-Sertoli cell) boundaries and found that incubation of cocultures in the continuous presence of NCAM antibodies caused release of essentially all gonocytes (but not Sertoli cells) from the monolayer. We also found, in (3) above, that gonocyte-Sertoli cell adhesion was very susceptible to phospholipase C in cocultures isolated from newborns and maintained in vitro for 2 hours or 1 day but not in cultures maintained for 3 days. Moreover, cells isolated from pups 5 days old were as resistant to enzyme treatment at 2 hours postplating as were cultures from newborns after 3 days in vitro. Thus, the way in which gonocytes adhere to Sertoli cells appears to change during the immediate postnatal period, as reflected by the observed change in phopholipase sensitivity, perhaps indicating production of a phospholipase C-resistant NCAM isoform by several days after birth. These data constitute new information on the way in which postnatal gonocytes adhere to Sertoli cells and provide a basis for future work in our ongoing exploration of germ cell development in the neonatal rat testis.

Localization of a spermatid-specific histone 2B protein in mouse spermiogenic cells.

Mammalian spermiogenesis is characterized by chromatin condensation and replacement of the histones typical of somatic and earlier spermatogenic cells by protamines in the nucleus. However, a spermatid-specific H2b histone (ssH2b) that has an unusual carboxyl-terminus containing a region rich in hydrophobic amino acids is transcribed and translated in mouse round spermatids. The hydrophobicity of this region suggested that the protein may be localized at the nuclear envelope, the initial site of chromatin condensation during spermiogenesis. To identify ssH2b in the spermatid nucleus, an antiserum (anti-ss-H2b127-138) was generated against a synthetic peptide corresponding to the unique carboxyl-terminus of the protein. Immunocytochemistry of fixed, frozen testicular sections at both the light and electron microscopic levels indicated that ssH2b is present in nuclei of round spermatids. Moreover, the protein was not found to be preferentially associated with the nuclear envelope, indicating that it is not involved in chromatin-nuclear envelope interaction in the round spermatid. Rather, it appeared to be distributed uniformly throughout the nucleus with the exception of its exclusion from the nucleolus. In addition, the ssH2b protein was not found in mature sperm even when the chromatin was partially decondensed, suggesting that it is present and functions only during a restricted period of spermatogenic development.

Gonocytes of male rats resume migratory activity postnatally.

In the testis of the neonatal rat, maturation of germ cells, or gonocytes, lays the foundations for spermatogenesis which will begin later in postnatal development. One of the most critical and yet least understood of the events that occur during the immediate neonatal period is relocation of gonocytes from the more central part of the seminiferous cord, where they are surrounded by Sertoli cells, to its periphery, where they contact the basement membrane. For the current study, we examined this change in gonocyte position by identifying some of the cellular mechanism involved, with the aim of determining whether movement of gonocytes to the basement membrane in vivo and development of cellular processes by these cells in vitro represents a resumption of migratory activity similar to that displayed by their fetal ancestors and by other motile cells. First, we used either thiamine pyrophosphatase cytochemistry or the fluorescent probe nitrobenzoxadiazole ceramide to visualize the Golgi complex in gonocytes and found that (1) this organelle matures and apparently enlarges in vivo with a time course paralleling movement of gonocytes to the basement membrane and undergoes similar changes in vitro that correlate with gonocyte process formation, and (2) the Golgi complex is located in perinuclear cytoplasm facing the apparent direction of gonocyte movement in vivo and in cytoplasm near the cellular process in the great majority of elongated gonocytes in coculture. Next we used two drugs, brefeldin A and monensin, which have in common their ability to disrupt the Golgi complex, and found that both drugs prevent process formation by gonocytes in a manner that is completely reversible. We also tested the involvement of the cytoskeleton in gonocyte elongation by utilizing nocodazole to disrupt and taxol to stabilize microtubules, as verified by alpha-tubulin immunofluorescence. Inclusion of the drug abolished (taxol) or substantially diminished (nocodazole) the ability of gonocytes to elongate in a reversible manner. We also found that the Golgi complex was intact in the presence of taxol and that microtubules were intact in the presence of both Golgi complex-specific drugs. Thus, our findings indicate that (1) both the Golgi complex and microtubules are involved in development of processes by gonocytes and (2) neither structure is sufficient by itself to allow these cells to elongate. Taken together, our data provide new evidence suggesting that the cellular mechanism utilized by postnatal gonocytes in relocating to the basement membrane are those mediating active migration.(ABSTRACT TRUNCATED AT 400 WORDS)

Reinitiation of gonocyte mitosis and movement of gonocytes to the basement membrane in testes of newborn rats in vivo and in vitro.

Movement of postnatal gonocytes to the periphery of the seminiferous cord, where they contact the basement membrane, and resumption of mitosis by these previously quiescent cells are likely to be critically important in establishing spermatogenesis in neonatal rats. We used several approaches both in vivo and in vitro to determine precisely when each of these two events begins, to study their temporal relationship to each other, to determine whether gonocyte division is a prerequisite for relocation or vice versa, and to probe the source of factors initiating and/or regulating these events. Both light and electron microscopy were used to determine that the first gonocytes make contact with the basement membrane on postnatal day 4, while quantitative autoradiography following 3H-thymidine administration in vivo indicated that the first gonocytes to re-initiate cell division do so one day earlier, on day 3, and that the percentage of gonocytes dividing remains at a stable level through day 5. Moreover, we organ-cultured neonatal testes from birth onwards in the presence of defined, serum- and hormone-free medium and determined that both proliferation and relocation of gonocytes begin and continue in vitro as in vivo. This observation argues against involvement of extratesticular factors in stimulating gonocyte relocation and division, and points to the testis itself as the most likely source of agent(s) regulating postnatal maturation of these cells. In other, similar incubations we included 3H-thymidine for varying periods of time to label either those gonocytes that are the first to divide or all gonocytes that divide during the first 48 hr of culture. From these studies, we confirmed that the first gonocytes to divide do so while separated from the basement membrane and found that, although some cells divide before moving peripherally, others do not.

Neonatal gonocytes co-cultured with Sertoli cells on a laminin-containing matrix resume mitosis and elongate.

We co-cultured gonocytes and Sertoli cells isolated on the day of birth and observed the appearance, 1 and 3 days after the start of culture, of gonocytes that had developed cellular processes and that were labeled by [3H]thymidine, respectively. These events occurred in the absence of hormones, etc. and with a time course very similar to that seen in vivo. In other incubations, we found that the presence of laminin in the underlying substrate was critical in promoting proliferation and elongation of gonocytes. These observations strongly suggest that interactions between gonocytes and other testicular cells/factors in the co-cultures promote maturation of these germ cells in vitro, and thus provide new evidence to support the concept that paracrine mechanisms are important during testicular development as well as in adults.

Functional coupling of neonatal rat Sertoli cells and gonocytes in coculture.

Interaction between Sertoli cells and germ cells is likely to be critical for normal development of the testis. We have established and characterized cocultures of neonatal Sertoli cells and gonocytes and have begun to study the physical and functional relationship between these cells in vitro. Cells were isolated from rat pups by sequential enzymatic treatment and cultured in serum-free medium. When plated on Matrigel, Sertoli cells rapidly attach, and gonocytes adhere to the underlying Sertoli cells shortly thereafter. We observed that some of these germ cells develop cytoplasmic processes and elongate during the first day of culture, essentially mimicking their behavior in vivo. Electron microscopic examination of typical cultures revealed the presence of desmosome-like adhesion sites and apparent gap junctions between Sertoli cells and gonocytes. To determine whether Sertoli cells and gonocytes are functionally coupled in the cocultures, we used the glass bead-loading technique of McNeil and Warder to introduce Lucifer yellow (LY), a gap junction-permeant probe, and Rhodamine-dextran (RD), a larger marker excluded by gap junctions, simultaneously into cultures 24 h after plating. Immediate fixation and viewing of cultures with fluorescence microscopy indicated that all bead-loaded cells received both probes. We studied other living cultures 10 min after bead-loading and located RD-negative (i.e. nonbead-loaded) gonocytes that were in obvious contact with RD- and LY-positive bead-loaded Sertoli cells; these gonocytes were scored for the presence or absence of cytoplasmic LY. This analysis revealed that many gonocytes were able to obtain LY from adjacent Sertoli cells, presumably via gap junctions maintained with these cells. In addition, we quantified the percentage of gonocytes that elongated with increasing time in vitro and correlated the morphology of these cells with their ability to acquire LY from adjacent Sertoli cells. Our findings indicate that although the absolute numbers of gonocytes present decreases, more of those remaining elongate as time in vitro increases. We can also conclude from our data that gonocytes with and without processes are equally likely to be coupled with Sertoli cells under these conditions. These observations provide the first demonstration of functional coupling between Sertoli cells and premeiotic germ cells. Together with our morphological observations, they suggest that gap junction-mediated communication between these cells may be involved in stimulating or regulating changes in the gonocyte population during postnatal development of the testis.

Naltrexone normalizes the suppression but not the surge of delta 5-3 beta-hydroxysteroid dehydrogenase activity in Leydig cells of stressed rat fetuses.

Rat fetuses from mothers stressed chronically by immobilization and high intensity illumination beginning on day 14 of gestation have higher than normal levels of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity in Leydig cells on day 17 of gestation and lower than normal levels on days 18 and 19. Plasma testosterone titers in normal and stressed male fetuses closely parallel the activity of 3 beta HSD in fetal Leydig cells. In the present study quantitative cytochemistry was used to determine whether the stress-induced alterations in 3 beta HSD activity could be prevented by treating the mother with naltrexone, an opioid receptor blocker, before each stress session. Naltrexone normalized 3 beta HSD activity on days 18 and 19 of gestation, suggesting that the stress-induced suppression involves the endogenous opioid system. In contrast, naltrexone did not prevent the elevation in enzyme activity seen on day 17 in stressed fetuses. The persistence of a stress-induced surge on day 17, in spite of naltrexone therapy, suggests that some nonopioid mechanism is operational at that time.

Endorphin suppresses FSH-stimulated proliferation of isolated neonatal Sertoli cells by a pertussis toxin-sensitive mechanism.

During perinatal development, when the size of the Sertoli cell population is determined, Leydig cells produce beta-endorphin, a peptide which may interact with Sertoli cells to modify their FSH-responsiveness, as suggested by our previous work. The goal of the present study was first, to test directly the possibility that beta-endorphin modifies the proliferative response of neonatal Sertoli cells to FSH, and second, to gain information on a mechanism(s) involved in any observed effect. We treated isolated 6-day-old Sertoli cells with FSH or vehicle in vitro and measured their incorporation of exogenous, radiolabeled thymidine with quantitative autoradiography. After 2 days in culture with FSH, we detected a 10-fold increase in the rate of Sertoli cell proliferation. The level of cell division in these FSH-treated cultures was identical to that in other cultures exposed to cAMP under similar conditions. In addition, inclusion of beta-endorphin 3 hr prior to FSH or cAMP decreased the effect of the hormone by 50% but left the cAMP response unchanged. Thus, beta-endorphin acts on isolated, neonatal Sertoli cells at a point prior to intracellular production of cAMP to suppress their response to FSH. When other cultures were treated with pertussis toxin, a blocker of intracellular GTP-binding proteins such as Gi, before sequential addition of endorphin and FSH, the effect of beta-endorphin on FSH-responsiveness was abolished. Moreover, when other cultures were exposed to pertussis toxin in the absence of endorphin, followed by FSH, their response to the hormone was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)