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Background/aim mmune checkpoint inhibition (CPI) has an increasing impact in the multimodal treatment of locally advanced non-small cell lung cancer (LA-NSCLC). Increasing evidence suggests treatment outcome depending on tumor cell PD-L1 expression. The purpose of this retrospective study was to investigate the prognostic value of PD-L1 expression on tumor cells in combination with CD8+ tumor stroma-infiltrating lymphocyte (TIL) density in inoperable LA-NSCLC treated with concurrent chemoradiotherapy (CRT). Patients and method We retrospectively assessed clinical characteristics and initial tumor biopsy samples of 31 inoperable LA-NSCLC patients treated with concurrent CRT. Prognostic impact of tumor cell PD-L1 expression (0% versus ≥1%) and CD8+ TIL density (0–40% vs. 41–100%) for local control, progression-free (PFS) and overall survival (OS) as well as correlations with clinicopathological features were evaluated. Results Median OS was 14 months (range: 3–167 months). The OS rates at 1- and 2 years were 68 and 20%. Local control of the entire cohort at 1 and 2 years were 74 and 61%. Median PFS, 1-year and 2-year PFS were 13 ± 1.4 months, 58 and 19%. PD-L1 expression neg/CD8low) and type IV (PD-L1pos/CD8low) tumors (median OS: 57 ± 37 vs. 10 ± 5 months, p = 0.05), respectively. Conclusion Assessment of PD-L1 expression on tumor cells in combination with CD8+ TIL density can be a predictive biomarker in patients with inoperable LA-NSCLC treated with concurrent CRT.

Serine/threonine protein phosphatase 1 (PP1) consists of four ubiquitously expressed major isoforms, two of which, PP1gamma1 and PP1gamma2, are derived by alternative splicing of a single gene, Ppp1cc. PP1gamma2 is the most abundant isoform in the testis, and is a key regulator of sperm motility. Targeted disruption of the Ppp1cc gene causes male infertility in mice due to impaired spermiogenesis. This study was undertaken to determine the expression patterns of specific PP1 isoforms in testes of wild-type mice and to establish how the defects produced in Ppp1cc-null developing sperm are related to the loss of PP1gamma isoform expression. We observed that PP1gamma2 was prominently expressed in the cytoplasm of secondary spermatocytes and round spermatids as well as in elongating spermatids and testicular and epididymal spermatozoa, whereas its expression was weak or absent in spermatogonia, pachytene spermatocytes, and interstitial cells. In contrast, a high level of PP1gamma1 expression was observed in interstitial cells, whereas much weaker expression was observed in all stages of spermatogenesis. Another PP1 isoform, PP1alpha, was predominant in spermatogonia, pachytene spermatocytes, and interstitial cells. Examining the temporal expression of PP1 enzymes in testes revealed a striking postnatal increase in PP1gamma2 levels compared with other isoforms. Testicular sperm tails from Ppp1cc-null mice showed malformed mitochondrial sheaths and extra outer dense fibers in both the middle and principal pieces. These data suggest that in addition to its previously documented role in motility, PP1gamma2 is involved in sperm tail morphogenesis.

OBJECTIVE. We describe the significance of detecting focal areas of hypermetabolism in the breast in patients undergoing PET/CT for reasons other than for breast cancer detection or staging.CONCLUSION. When evaluated, almost all of the abnormal foci detected in the breast subsequently proved to be breast carcinoma, specifically infiltrating ductal carcinoma.

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 NC...

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.

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.

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.

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.

The induction of tumor cell death is one of the major goals of radiotherapy and has been considered to be the central determinant of its therapeutic outcome for a long time. However, accumulating evidence suggests that the success of radiotherapy does not only derive from direct cytotoxic effects on the tumor cells alone, but instead might also depend – at least in part – on innate as well as adaptive immune responses, which can particularly target tumor cells that survive local irradiation. The clearance of dying tumor cells by phagocytic cells of the innate immune system represents a crucial step in this scenario. Dendritic cells and macrophages, which engulf, process and present dying tumor cell material to adaptive immune cells, can trigger, skew, or inhibit adaptive immune responses, respectively. In this review we summarize the current knowledge of different forms of cell death induced by ionizing radiation, the multi-step process of dying cell clearance, and its immunological consequences with special regard toward the potential exploitation of these mechanisms for the improvement of tumor radiotherapy.

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.

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.

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.

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.

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)

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.

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.

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.

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.

Our understanding of spermatogenesis in the adult testis has increased substantially in past years, as more and more investigators have focused on the role of interactions between somatic and germ cells in supporting this process. We now recognize, for example, the way in which adhesive interactions between Sertoli and germ cells control movement of young spermatocytes from the basal into the adluminal compartment and the critical support provided to the adluminal cohort of spermatogenic cells by the Sertoli cells. Considerably less is known, however, about how spermatogonia begin their passage through spermatogenesis and even less is understood about the ancestors of these cells, the gonocytes, and how they mature during the prespermatogenic period of testicular development.

The Myb gene family currently consists of three members, named A-, B- and c-myb1,2. These genes encode nuclear proteins that bind DNA in a sequence-specific manner and function as regulators of transcription. In adult male mice, A-myb is expressed predominantly in male germ cells2,3. In female mice, A-myb is expressed in breast ductal epithelium, mainly during pregnancy-induced ductal branching and alveolar development. We report here that mice homozygous for a germline mutation in A-myb develop to term but show defects in growth after birth and male infertility due to a block in spermatogenesis. Morphological examination of the testes of A-myb−/− males revealed that the germ cells enter meiotic prophase and arrest at pachytene. In adult homozygous null A-myb female mice, the breast epithelial compartment showed underdevelopment of breast tissue following pregnancy and the female mice were unable to nurse their newborn pups. These results demonstrate that A-myb plays a critical role in spermatogenesis and mammary gland development.

Sperm production in the mature testis is the result of complex processes that have been the focus of intense study in recent years, resulting in an increased understanding of the mechanisms underlying spermatogenesis. However, far less information is available concerning the early postnatal development of the testis when a number of events occur that are critical for the later onset of spermatogenesis, including the resumption of germ cell mitosis and the arrival of some of these cells at the basement membrane.

A study of the efficiency of space closure after premolar extraction was undertaken, comparing a nickel-titanium closed coil spring and an elastic retraction module by using sliding mechanics along an 0.019 x 0.025-inch stainless steel arch wire in 0.022 x 0.028-inch preadjusted stainless steel brackets. The rate of space closure in 17 subjects was analyzed from study models and was found to be significantly greater and more consistent with the nickel-titanium closed coil springs than with the elastic modules, in both arches. There were no clinically observable differences in the tooth positions between the respective techniques.

Introduction: Studies of FNP in the diagnosis of focal lesions of the pancreas have reported a sensitivity of 57-93% and a specificity of 95-100% for the method. The objective of the present investigation was to evaluate the sensitivity and specificity of FNP in the diagnosis of focal lesions of the pancreas. Patients and Methods: Between May, 1995 and June, 1996 we performed FNP on 25 patients (14 females, 11 males; average age 61.4 + 10.91 years, range 31-77 years). In nine cases, confirmation of findings was surgical, in the remaining 16 patients based on a one-year clinical follow-up period. Results: Aspiration of diagnostically adequate material was possible in 18 cases (72%) (malignancy, 10; chronic pancreatitis, 4; benign lesions, 4). For the other seven patients (28%), clinical follow-up revealed pancreatic carcinoma in two patients, pancreatitis in four patients, while in one case even the surgical specimen obtained for histologic examination failed to provide a diagnosis. For patients with adequate cytology findings, one-year follow-up revealed false-negative findings in three cases: three of four patients with chronic pancreatitis developed carcinoma. Discussion.: FNP in the diagnosis of focal pancreatic lesions showed a sensitivity of 77% and a specificity of 100%. There were no complications observed in our group. Conclusion: Negative findings at FNP in chronic pancreatitis does not exclude the possibility of carcinoma. In the case of equivocal findings, criteria for surgery should be broad.

Quantitative cytochemistry was used to determine the effect of subjecting pregnant rats to environmental stress on the activity of delta 5-3 beta hydroxysteroid dehydrogenase (3 beta-HSD) in Leydig cells of their fetuses. Enzyme activity was measured by microspectrophotometry in individual Leydig cells in cryostat sections of fetal testes on Days 16-21 postconception. Fetuses of stressed mothers lacked the peak of enzyme activity on Days 18 and 19 of gestation that is characteristic of Leydig cells of normal fetuses at this time. In addition, both before and after these 2 days, 3 beta-HSD activity in Leydig cells of stressed fetuses was significantly higher than normal. The altered developmental pattern of 3 beta-HSD activity in the stressed fetuses largely corresponds to the changes in plasma testosterone found previously in male fetuses of mothers exposed to the same regimen of stress. Thus, in the fetal Leydig cell, the activity of 3 beta-HSD, a key steroidogenic enzyme, can be modified by environmental stress, and provides an index of steroidogenic activity of the fetal testes and of the titers of circulating testosterone.

Proliferation of Sertoli cells in the rat testis occurs only during the perinatal period and is maximal during fetal life. This interval is thus of critical importance in establishing the complement of Sertoli cells that populates the adult testis. FSH has been implicated in this process, but direct evidence in support of its involvement is lacking. In the present study, we have used in vivo and in vitro approaches to determine whether FSH produced by the fetal pituitary has a role in regulating Sertoli cell division in the fetal testis of the rat. On day 18 of gestation, just before the onset of maximal Sertoli cell proliferation, fetuses were either decapitated in utero or given antiserum to FSH. Light microscope autoradiography was then used to compare uptake of [3H]thymidine by Sertoli cell nuclei in testes from decapitated or antiserum-treated fetuses to that in corresponding controls on the following day. Both treatments produced dramatic and equal reductions in the percentages of Sertoli cells preparing to divide on day 19, suggesting that FSH from the fetal pituitary stimulates Sertoli cell proliferation in fetal testes. The effect of FSH or (Bu)2cAMP on Sertoli cell proliferation was also studied in vitro by placing testes from intact or decapitated fetuses into organ culture, with or without exogenous hormone or cyclic nucleotide. In all cases, [3H]thymidine was present for the final 4 h of culture. When testes were placed into medium containing isotope immediately after their removal from the fetus, the difference in labeling between testes from intact and decapitated fetuses was similar to that measured in vivo. After testes from decapitated fetuses were cultured for 8 h with or without FSH or (Bu)2cAMP, labeling of Sertoli cells in the treated group increased markedly over that in untreated cultures. After 28 h of exposure to FSH or (Bu)2cAMP, labeling in testes from decapitated fetuses remained significantly higher than that in corresponding untreated controls. In contrast, when testes from intact rats were cultured for 8 h in the presence of either cAMP or FSH, (Bu)2cAMP, but not FSH, brought about an increase in the percentage of Sertoli cells labeled compared to the control value. However, after exposing these testes to either FSH or (Bu)2cAMP for 28 h, the percentage of Sertoli cells labeled was greatly enhanced. Taken together, the data obtained from these experiments identify FSH as a major factor in controlling expansion of the Sertoli cell population during fetal development of the rat.(ABSTRACT TRUNCATED AT 400 WORDS)

In a previous study, we used autoradiography to demonstrate specific FSH-binding sites on Sertoli plasma membranes in the basal compartment of rat seminiferous tubules. The present study was undertaken to determine whether spermatogonia in the rat testis also possess FSH-binding sites. Approximately 1 Mg [l25I]hFSH was administered as an intratesticular injection to 40-day-old rats that had been hypophysectomized 10 days earlier; a second group of rats received a 500-fold excess of cold hormone along with the [I25I]- hFSH, and served as a control for specificity of binding. Subsequently, all testes were perfused with a hyperosmotic fixative, which produces preferential shrinkage of spermatogonia and Sertoli cells in the basal compartment. The resulting separation of the cells from one another allowed us to determine whether spermatogonia were the source of any autoradiographic grains. In the absence of excess cold hormone, some grains were clearly localized to spermatogonial surfaces, although the majorit...

Previous evidence has indicated that the testicular receptors for FSH reside on plasma membranes in seminiferous tubules, and that the Sertoli cell is a primary target of the hormone. Since the Sertoli cell is large and elaborately structured, it is important to know whether there is a regional distribution of receptors over its extensive surface. In the present study, the specific binding of 25I-labeled hFSH to seminiferous tubules of hypophysectomized rats was examined, utilizing autoradiography at both the light and electron microscope levels. Purified hFSH (LER-1575C) was iodinated to an average specific activity of 29 μCi/μg, and approximately 1 μg was administered intratesticularly in 40 day old rats which had been hypophysectomized 10 days earlier. In some cases, testosterone was administered after hypophysectomy in order to maintain spermatogenesis. Controls were of two types: 40 day old intact littermates in which endogenous FSH presumably occupied most of the receptors, and hypophysectomized rat...

Rat pups were hemicastrated and uptake of [3 H] thymidmne by Sertoli cells in the remaining testis was compared to that in testes of sham-operated pups at intervals of from 8 h to 21 days after surgery. Labeled thymidine was administered subcutaneously 2 h before sacrifice. Testes were processed for light microscope autoradiography and the percent of Sertoli cell nuclei that had incorporated [3 H] thymidmne was determined by scoring nuclei in tissue sections as labeled or unlabeled. The percentage of cells labeled was increased in hemicastrates over intact controls by 8 h after surgery and testicular hypertrophy became apparent in hemicastrates by the following day. Labeling of Sertoli cells in hemicastrates remained elevated for 4 days and then returned to normal. When plasma levels of gonadotropmns were measured in both groups 4 days after surgery, follicle-stimulating hormone (FSH) was found to be more than twice normal in hemicastrates while luteinizing hormone (LH) was unchanged. The effect of testosterone on the response of Sertoli cells to hemicastration was also examined. In hemicastrates, 2 days of androgen therapy depressed, and an additional 2 days abolished, the proliferative response of the Sertoli cells. Our findings suggest that increased proliferation of Sertoli cells within the remaining testis is involved in the enlargement of the testis that follows hemicastration. They also imply that prevention of compensatory hypertrophy by testosterone involves interference with this response of Sertoli cells in some way. Finally, our data implicate FSH in control of Sertoli cell proliferation in vivo in immature rats.

The pituitary-testicular axis was investigated in the streptozotocin diabetic male rat to determine the relationship between hormonal alterations and Leydig cell morphology. Male Wistar rats (60 days of age) were made diabetic with a single intravenous injection (65 mg/kg body weight) of streptozotocin and were studied with non-diabetic controls at 7–11, 14, 21, and 28 days after treatment. The observations on these animals were compared to those from diabetic rats (28 days) treated with 1–2 U protamine zinc insulin/100 mg body weight. Rats with untreated diabetes had hyperglycaemia and weighed less than controls. Untreated rats also had low plasma testosterone levels associated with decreased prostate and seminal vesicle weights. Insulin treatment partially or completely prevented these abnormalities. Studies of testosterone production y perfused testis suggested Leydig cell insensitivity to LH as one possible cause of decreased androgen blood levels in diabetic rats. Lipid droplets not found in Leydig cells of normal rats were seen in diabetic animals. Plasma FSH levels were also low in untreated diabetic rats but were normal in insulin

Neonatal development of the rat testis involves a number of critical events including re-entry of gonocytes into the cell cycle and eventual loss of many of these cells and their progeny via apoptosis. Since surviving gonocytes give rise to subsequent generations of germ cells, regulation of their fate is critical for adult testicular function. Here, we have identified a role for short-type PB-cadherin (STPB-C) in promoting survival of gonocytes in neonatal rats and we have linked its expression to the JAK-STAT signaling pathway. These findings were obtained with varied approaches including use of transgenic rats overexpressing STPB-C which were studied with protein microarrays and other techniques, direct examination of germ cell apoptosis and survival in gonocyte–Sertoli cell co-cultures, and direct study of the JAK-STAT pathway in these models and in L cells transfected with STPB-C. These data provide new information on the regulation of gonocyte fate and exciting new evidence supporting a link between the JAK-STAT pathway and cadherin-based cell–cell interactions.

To probe the possible role of endogenous opiates in Sertoli cell proliferation during testicular development, the effect of interfering with beta-endorphin action either in vivo or in vitro was determined. The percent of Sertoli cells dividing was measured with quantitative autoradiography in [methyl 3H]-thymidine-exposed fetal testes maintained in organ culture with or without FSH, in the presence or absence of the opiate blocker naloxone. After 1 or 2 days in culture, naloxone enhanced the rise in Sertoli cell proliferation seen with FSH alone, while 2 days of incubation with naloxone alone markedly raised the percent of Sertoli cells dividing above that in untreated cultures. Moreover, when endorphin antiserum was injected directly into testes of pups and Sertoli cell proliferation in vivo measured 8 or 19 h later, there was a dramatic increase in the percent of Sertoli nuclei labeled by [methyl 3H]-thymidine compared to controls. These findings suggest that beta-endorphin produced within the testis is a paracrine modifier of the proliferative response of Sertoli cells to FSH. This implies that communication occurs between Leydig and Sertoli cells during development via endogenous testicular opiates.

To probe the relationship between the size of the Sertoli cell population, established during perinatal development, and production of germ cells in the adult testis, a Sertoli cell-depleted rat model was developed. This was accomplished by delivering an antimitotic drug, cytosine arabinoside (araC), directly to the testis of newborn pups. Initial studies of these araC-treated neonates indicated that 1) the drug is cleared rapidly from the testis; 2) it substantially reduces the level of Sertoli cell proliferation; 3) Sertoli cell division ceases at a normal time in spite of the previous drug treatment; and 4) araC itself has no residual effect on germ cell proliferation, which begins several days after the injection. Pups given araC were allowed to reach maturity, and their testes were perfuse-fixed for light microscopic morphometry. When the numbers of Sertoli cells in adult rats given araC as were compared with those in normal littermates, a 54% decrease in the size of the Sertoli cell population was detected in treated rats, now referred to as Sertoli cell-depleted. Moreover, when round spermatids were quantified and compared in normal and Sertoli cell-depleted adults, testes of the latter were found to contain 55% fewer round spermatids. Since, in the araC-treated group, the decrease in Sertoli cell population size was paralleled by a reduction in spermatid production of equal magnitude, the number of round spermatids per Sertoli cell was essentially identical in normal and Sertoli cell-depleted animals. Measurements of serum androgen-binding protein (ABP) and FSH in both groups indicated that the circulating level of ABP in Sertoli cell-depleted rats was approximately half, and the concentration of FSH approximately twice, that in normal animals. Thus, even though FSH is elevated in Sertoli cell-depleted rats, the production of ABP per Sertoli cell is unchanged. In addition, collective volume of Leydig cells and ventral prostate weights were normal in the Sertoli cell-depleted group, suggesting that Leydig cell function in these rats is normal. In summary, a Sertoli cell-depleted rat model has been produced by interfering specifically with Sertoli cell proliferation early in postnatal life, before onset of germ cell division. Moreover, our findings with this model indicate that production of normal numbers of germ cells in adults depends, at least in part, on the size of the Sertoli cell population. Thus, our observations identify the perinatal period, when the Sertoli cell population is established, as critical for development of quantitatively normal spermatogenesis in the adult.

Proliferation of Sertoli cells during fetal and postnatal development of the rat was examined and quantified with light microscope autoradiography. Fetuses in utero were injected subcutaneously with 3H-thymidine. The percentages of Sertoli nuclei that had incorporated label were determined in autoradiographs from fetuses aged 16 through 21 days of gestation. To compare the degree of Sertoli cell proliferation during fetal development with that occurring after birth, pups were also studied at intervals between the day of birth and 3 weeks of age. For each fetus or pup, at least 500 Sertoli cell nuclei in each of three sections were scored as labeled or unlabeled. These data were subjected to analysis of variance and the Newman-Keuls test. The percentage of Sertoli cells incorporating 3H-thymidine increased progressively from day 16 of gestation onward, to a maximum of 26.8% on day 20, two days before birth. Thereafter, this percentage dropped steadily until, in pups 21 days after birth, no labeled Sertoli cells were detected. These findings highlight the fetal period as the time of greatest expansion of the Sertoli cell population and indicate that, at birth, proliferation of these cells is already on the decline.

Hyperglycemia (experimental diabetes) was induced in adult male rats by destruction of the pancreatic beta cells with a single intravenous injection of streptozotocin (STZ). Testes from diabetic, from insulin-treated diabetic, and from sham-injected normal rats were fixed by vascular perfusion. The fine structure of Leydig cells was examined at two, three, and four weeks after the STZ injection in the untreated diabetic animals, and at four weeks in the controls and insulin-treated diabetic rats. A number of morphological changes was observed in Leydig cells of untreated diabetic animals. Most obvious of these was an accumulation of lipid droplets, not normally present in Leydig cells in adults of this species. Smooth endoplasmic reticulum (SER) was markedly reduced in Leydig cells of the hyperglycemic rats. Several types of intracellular bodies were seen exclusively in Leydig cells of the untreated diabetic animals. Many resembled secondary lysosomes or dense bodies, while others appeared to be autophagic vacuoles. In addition, a small, granule-containing lamellar structure was seen either within a typical dense body or free in the cytoplasm. Myelin-like structures were commonly observed within the cytoplasm of the Leydig cell or within mitochondria. The appearance of the mitochondria in diabetic rats was otherwise normal. The extracellular spaces surrounding Leydig cells from untreated hyperglycemic rats also contained large accumulations of myelin-like material. These structural changes appear to be direct consequences of the diabetic state of the animals, since the ultrastructure of insulin-treated diabetic rats did not differ from that of the controls. These findings may reflect an alteration or breakdown of Leydig cell components normally involved in the synthesis of androgen, and correlate with previous reports of lowered circulating levels of testosterone in diabetic rats.

It is now well established that follicle-stimulating hormone (FSH) acts primarily on the seminiferous tubule of the mammalian testis, and that receptors for this action are located on tubule plasma membranes. Specific FSH-binding sites have been demonstrated in isolated seminiferous tubules from immature and mature rats, and it has been shown that most of the binding activity resides in the plasma membrane fraction (Means and Vaitukaitis, 1972; Means, 1973). The biochemical events that are initiated by the binding of FSH to its receptor are also well characterized. Incubation of FSH with either seminiferous tubules (Dorrington et al., 1972) or plasma membranes isolated from tubules (Means, 1975) results in an elevation of cyclic AMP (cAMP) levels that is hormone-and tissue-specific. The cAMP seems to act as an intracellular messenger, causing activation of cAMP-dependent protein kinase and subsequent transcriptional and translational events (Means, 1975).