STUDY QUESTION Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? SUMMARY ANSWER We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. WHAT IS KNOWN ALREADY Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in vitro or testosterone production was observed. LIMITATIONS, REASONS FOR CAUTION The human testis is very different in physiology from the rat testis, further investigations are still needed to optimize the organ culture system for future use in humans. WIDER IMPLICATIONS OF THE FINDINGS The successful differentiation of undifferentiated spermatogonia using the testis explant culture system might be employed in future to produce sperm from human spermatogonia as a clinical tool for fertility preservation in boys and men suffering infertility. LARGE SCALE DATA None. STUDY FUNDING AND COMPETING INTEREST(S) This work was supported financially by the Frimurare Barnhuset in Stockholm, the Paediatric Research Foundation, Jeanssons Foundation, Sällskåpet Barnåvard in Stockholm, Swedish Research Council/Academy of Finland, Emil and Wera Cornells Foundation, Samariten Foundation, the Swedish Childhood Cancer Foundation as well as through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet. All authors declare no conflicts of interests.

The toxicologic literature abounds with examples of drugs and environmental chemicals that cause changes in spermatogenesis and/or epididymal sperm in laboratory animals. Unless proven otherwise, the assumption must be made that similar changes are likely to occur in humans exposed to these chemicals. For those working in the pharmaceutical industry, this can mean the costly termination of a drug development program and the loss of a potentially valuable therapeutic molecule unless studies can be conducted to investigate potential mechanisms of toxicity and/or realistically predict human risk. For those working in regulatory environmental toxicity, mechanistic studies are rarely conducted but demonstration of a mode of action (MOA) can provide valuable information to allow a better understanding of the real threat of environmental toxicants versus the perceived threat. For both disciplines, it is essential to understand the basic science underlying spermatogenesis and sperm maturation in order to address any mechanistic approach to investigating a toxicologic lesion. The toxicologic pathologist is generally the person to first identify and sound the alarm regarding a male reproductive toxicant in a drug development or environmental chemical program. Organ weights and histopathology will be the main (often only) information that will be available from repeat dose studies. Given a good understanding of spermatogenesis and spermatogenic staging, the pathologist may be able to take a guess at the earliest cell type affected (Sertoli or germ cell), get a feel for the pathogenesis and recovery of the lesion over time, and they may even be able to assess whether endocrine disturbance is a major and primary event. Very soon after sounding the alarm, a lot of questions will be asked regarding the likely mechanism of toxicity, whether the spermatogenic disruption is “on or off target” for the therapeutic molecule, and is the toxicity, (which often affects one species and not the other) relevant to man? Such questions are extremely difficult to answer or even address when dealing with disturbances of spermatogenesis because of the complexity of the cellular interactions within the testis, the relative lack of knowledge of the physiology and molecular biology of spermatogenesis and the fact that we are often working with species (e.g. dog and monkey) for which there is remarkably little basic biologic information. In fact, many of these questions are never addressed by pharma companies with a reproductive issue, partly because of time and money constraints, but also because the main objective of repeat dose regulatory studies is risk assessment. So if any follow up mechanistic work is conducted, it is limited and only aimed at whether an effect is relevant to man or justifying why a higher dose can safely be used in clinical trials. This approach contrasts with basic research on cell physiology, endocrinology and molecular biology of spermatogenesis where individual processes are studied in great depth and where occasionally, chemicals may be used to disrupt a process. The objective of this special issue is to try and bring regulatory toxicology and academic, investigative toxicology together to provide relevant information that can benefit both academic and toxicologic-driven disciplines. Specifically, the authors of the main chapters here have been charged with explaining the biology underlying their topic. When we see a lesion, what is going on in the cells to produce that kind of lesion? The intent is to provide a quick path to understanding, and access to relevant literature. This Special Issue begins with a pictorial review of the broad types of changes that confront the pharmaceutical toxicologic pathologist on a daily basis. Morphologic manifestations of testicular and epididymal toxicity by Justin Vidal and Katherine Whitney provides an atlas and review of the typical features that lead the pathologist to draw conclusions regarding the main cell type injured, possible subcellular targets and the progression of a lesion from its subtle early features through to the non-specific endstages of tubular degeneration and tubular atrophy. This provides the backdrop for the subsequent detailed reviews from academic contributors explaining what might be expected (the Signature Lesion) when specific aspects of reproductive physiology are disturbed and the ways potential regulatory pathways could be disrupted to explain that lesion. This includes a detailed review of the cytoskeleton and the various critical functions that it regulates in Testicular histopathology associated with disruption of the Sertoli cell cytoskeleton by Kam Johnson, which details the roles of actin microfilaments, the intermediate filaments, and microtubules. The cytoskeleton is integral to the structural support of the seminiferous epithelium and the functioning of the multiplicity of cell junctions between Sertoli and germ cells and

BACKGROUND The U.S. EPA revised the Reproduction and Fertility Effects Test Guideline (OPPTS 870.3800/OECD 416) in 1998, adding numerous endpoints in an effort to incorporate new methodologies, improve the sensitivity for detecting reproductive toxicants, and more efficiently utilize study animals. Many of these new endpoints have not been used in regulatory reproductive toxicology studies prior to their inclusion in the test guidelines; thus, the Health and Environmental Sciences Institute (HESI) of the International Life Sciences Institute (ILSI) initiated the Reproductive Endpoints Project to examine the utility of these new endpoints. METHODS This report provides a retrospective analysis of 43 multi-generation studies (16 in Wistar rats, 27 in Sprague-Dawley rats) conducted according to the latest version of the test guidelines. It focuses on vehicle (negative) control values (means and ranges) for the various endpoints to examine inter-laboratory variability. RESULTS Based on the compiled data, the most variable endpoints across laboratories and their associated coefficients of variation (CV) for each generation were: percent abnormal sperm (166-205%), testicular spermatid concentration (126-147%), postimplantation loss (97-104%), primordial follicle counts (69%, only measured in P2 females), and epididymal sperm concentration (52-57%). Absolute and relative prostate and thymus weights, weanling uterine weights, and anogenital distance had CVs of 25-50%. Sources of variability included procedural differences between laboratories, inherent biological variability, and/or small sample sizes for some endpoints. CONCLUSIONS These inter-laboratory control data provide a means for laboratories to review their performance on reproductive toxicity measures, and provide perspective for interpreting their own control data and data from treated animals.

BACKGROUND: Given the role of nutrition and body weight gain in normal development, pharmaceuticals intended to reduce appetite and promote weight loss will generate safety data that may be challenging to interpret. To aid with this, the effects of feed restriction and subsequent body weight reductions on embryo-fetal development were investigated in the rat. METHODS: Groups of 20 timed pregnant female Sprague-Dawley rats were offered Certified Rodent Diet 5002 either ad libitum or in restricted amounts of 20, 15, 10, and 7.5 g/day from Gestation Day (GD) 6–17. Clinical signs, body weights, and food consumption were recorded. Cesarean sections were performed on GD 21 and fetuses were sexed, weighed, and examined for external, visceral, and skeletal development. RESULTS: Mean maternal body weights at the end of the feed restriction period, GD 18, were reduced 0.87 ×, 0.80 ×, 0.69 ×, and 0.63 × control mean in the 20, 15, 10, and 7.5g/day groups, respectively. Mean body weight gains for the restriction period inclusive, GD 6–18, were 0.49 × and 0.24 × control at 10 and 7.5 g/day, respectively, and a mean body weight loss occurred at 10 and 7.5 g/day (0.95 × and 0.85 × mean GD 6 body weight, respectively). Fetal body weights were reduced 0.95 ×, 0.93 ×, 0.90 ×, and 0.76 × control at 20, 15, 10, and 7.5 g/day, respectively. This resulted in a reduction in gravid uterine weight at 10 and 7.5 g/day. There were no external, visceral, or skeletal malformations attributed to feed restriction. There was an increase in the skeletal variation of wavy ribs and a decrease in ossification at 7.5 g/day. CONCLUSIONS: These data demonstrate that feed restriction-induced reductions in maternal gestational body weight gain of ∼50% compared to ab lib fed rats only caused a reduction in fetal body weight. Even up to a 15% maternal gestational body weight loss had no effect on embryo viability in rats, but retarded fetal growth significantly enough to induce minor changes in skeletal development. There were no external, visceral, or skeletal malformations associated with any of the levels of maternal body weight reduction or loss. Birth Defects Res B 2005. © 2005 Wiley-Liss, Inc.

BACKGROUND:Feed restriction with its resultant body weight loss impacts the rodent estrous cycle; however, the manifestation of these changes in a regulatory study design has not been documented. This study reports the effects of feed restriction in the context of an FDA regulatory submission. METHODS:Adult female rats (n = 20/group; weighing approximately 200 g each) were provided rodent chow ad lib (control) or at 20, 15, 10, or 7.5 g/rat/day (g/day) during a 2-week pre-mating phase, throughout the mating phase, and up to gestation day (GD) 7. On GD 8, all animals were provided ad lib feed until necropsy on GD 14. Estrous cyclicity, mating, and fertility parameters were evaluated. RESULTS:Ad lib rats consumed approximately 20 and 28 g/day during the pre-mating and gestation phases, respectively. All measured fertility parameters in the 20 g/day group were similar to control values. In the 15 g/day group, body weight was reduced by 16% at 2 weeks, prolonged diestrus occurred, and fertility was compromised due to reductions in corpora lutea. Within 2 weeks, mean body weight in groups receiving < or = 10 g/day was reduced by > or = 29% compared to ad lib values, and overt changes in estrous cyclicity, mating, and fertility occurred. The 7.5 g/day group was not sustainable beyond the pre-mating phase. CONCLUSIONS:For this study type, feed intake at < or = 50% ad lib values (< or = 10 g/day) was inadequate due to the magnitude and rapidity of body weight effects. Estrous parameters appeared slightly more sensitive than functional measures, as body weight changes of approximately 16% appeared near the threshold of changing routinely calculated estrous cycle parameters and were later associated with reduced fertility. In general, body weight differences of 10-15% by themselves were not adverse to normal reproduction (20 g/day).

BACKGROUND:Appropriate maternal nutrition and body weight gain during pregnancy is well established as a major factor in healthy prenatal development in humans. Given the role of nutrition and body weight gain in normal development, pharmaceuticals intended to reduce appetite and promote weight loss will generate developmental toxicity data that may be challenging to interpret. To aid with this, the effects of feed restriction, and subsequent reduction in maternal body weight gain, on embryo-fetal development was investigated in the rabbit.METHODS:Groups of 15 pregnant New Zealand White rabbits were offered 150 (control), 110, 75, 55, 35, and 15 g feed/day from gestation day (GD) 7-19. Cesarean sections were carried out on GD 29 and fetuses were examined for external, visceral, and skeletal development.RESULTS:Maternal body weights at the end of the feed restriction period (GD 20) were 0.97, 0.98, 0.93, 0.94, and 0.86 x control for the 110, 75, 55, 35, and 15 g feed/day groups, respectively. Only at 15 g feed/day was there a net maternal body weight loss (the GD 20 body weight was 0.93 x the GD 6 body weight) at the end of the feed restriction period. Six does aborted in the 15 g feed/day group; there were no other abortions associated with feed restriction. Fetal body weight was significantly reduced at 75, 55, 35, and 15 g feed/day (0.95, 0.90, 0.86, and 0.84 x control, respectively). There were no external or visceral malformations or variations, and no skeletal malformations associated with feed restriction. The incidence of fetuses with sternebrae 5 or 6 unossified was increased at feed levels < or = 75 g/day. At a feed level of 35 g/day there was an increase in unossified metatarsals and metacarpals, and an increase in the number of fetuses with a reduced number of caudal vertebrae ossified. Although these findings were not increased at a feed level of 15 g/day, the lack of dose response was likely due to increased abortion and subsequent decrease in fetuses available for evaluation at 15 g feed/day.CONCLUSION:These data demonstrate that feed restriction to feed levels that produce substantial reductions in maternal body weight gain can result in developmental toxicity expressed by abortion, reduced fetal weight, and alterations in ossification. Abortion only occurred when feed was restricted to an amount that produced maternal body weight loss (15 g feed/day) whereas reduced fetal weight and increased incidence of fetuses with unossified sternebrae, metatarsals, metacarpals, or caudal vertebrae were noted at feed levels of < or = 75 g/day. There were no fetal malformations associated with feed restriction.

The benefits and costs are discussed for the possibility of using pups generated in breeding studies for additional assessments, such as evaluating immunotoxicity. Such an approach is logistically challenging, but not overwhel ming.

Methoxyacetic acid (MAA) is a major metabolite of ethylene glycol monomethyl ether (EGME). Previous investigations of the testicular lesion induced by EGME have found that dividing meiotic cells are the most sensitive, although several stages of spermatocytes are also vulnerable. Preliminary data from this lab suggested the involvement of protein kinase activity in the development of this lesion, a hypothesis explored in the present studies. We used cultured seminiferous tubules (STs) from juvenile rats (25-day-old), exposed in vitro to MAA and several inhibitors of protein kinases. Nineteen h following a 5-h exposure to 5 mM MAA (the plasma level in vivo after a toxic dose of EGME), apoptotic spermatocytes were seen in early- and late-stage STs. Cell death was prevented by cotreatment with broad-spectrum inhibitors of protein kinases such as H-7, H-8, K-252a, W-7, and genistein. In corroboration, immunocytochemistry with antibodies to various kinases (PKCmu, zeta, and gamma, AKAP220, CaMKII, MLCK, and Src) showed increased staining around dying spermatocytes following EGME treatment in vivo. 2D-PAGE, autoradiography, and nanospray mass spectrometry was used to separate and identify proteins whose phosphorylation status was most greatly changed following exposure to MAA. One protein was identified by sequence analysis as being glucose-regulated protein 94 (grp94). Westem blotting and immunocytochemistry confirmed this finding. The data we present implicate kinase activities in the pathogenesis of this lesion and suggest the involvement of Sertoli cells.

The developing organism is considered to be more sensitive than the adult to immunotoxic agents. There is every reason, therefore, to include immune assessments in the regulatory testing for developmental toxicity of drugs that are intended to be used in young patients or pregnant woman. An effective strategy would be to incorporate immune assessments in the existing recommendations on pre- and post-natal toxicity study in the rat from the International Conference on Harmonisation. Immune assessments could also be included in juvenile toxicity studies to screen for effects resulting from post-natal exposure to the drug. Adequate testing methods are available to screen for developmental effects that result in immune depression. Routine immune assessments may comprise histopathological examination of the lymphoid organs/tissues and immunophenotyping of lymphocyte subsets in the blood, spleen, or thymus. These tests should be performed in rodents at various ages and at various stages of pre- and post-weaning development. Immunoglobulin and cytokine measurements, assessment of the T-cell dependent antigen response to sheep red blood cells or keyhole limpet haemocyanin antigens, and host resistance studies may be performed as apical tests at maturity. More research is required to develop methods for the detection of drugs that may render the developing organism more susceptible to hypersensitivity or autoimmunity.

Reproductive toxicity studies are increasingly including assessments of sperm parameters including motility, morphology, and counts. While these assessments can provide valuable information for the determination of potential reproductive toxicity, the methods for conducting the assessments have not been well developed in all laboratories and are continually evolving. The use of different methods in different laboratories makes comparison of data among laboratories difficult. To address the differences in methods, a working group was convened to discuss methods currently in use, share data, and try to reach consensus about optimal methods for assessing sperm parameters in rats, rabbits, and dogs. This article presents the consensus report, as well as future research needs, with the hope that optimized common methods will aid in the detection of reproductive effects and enhance interlaboratory comparisons.

Fumonisins are mycotoxins produced by Fusarium moniliforme and other Fusarium species. They are commonly found in corn and corn-based foodstuffs. Fumonisins inhibit sphingolipid (SL) biosynthesis, alter cellular SL profiles, and thus may affect cell proliferation and differentiation, both of which are important processes for reproduction. However, there are few reports of the effects of F. moniliforme or fumonisins on mammalian reproduction or development. To study the reproductive effects of this fungus, diets formulated with culture material of toxic F. moniliforme strain MRC 826 (CM) to provide 0, 1, 10, or 55 ppm fumonisin B1 (FB1) were fed to male and female rats beginning 9 and 2 weeks before mating, respectively, and continuing throughout mating, gestational, and lactational phases of the study. CM caused nephropathy, typical of FB1, in males fed > or = 10 ppm and females fed 55 ppm FB1. No significant reproductive effects were found in males (n = 12/group), dams, and fetuses examined on gestation day 15 (G15) (n > or = 8/group), or dams and litters through day 21 postpartum (n > or = 9/group). Litter weight gain in the 10 or 55 ppm FB1 groups was slightly decreased; however, gross litter weight and physical development of offspring were not affected. Altered SL ratios indicative of fumonisin exposure, specifically increased sphinganine to sphingosine ratios, were found in the livers of dams from the 55-ppm FB1 group on G15. However, SL ratios of abdominal slices, containing liver and kidney, of fetuses from control and high-dose litters did not differ. In a second experiment, two dams were injected intravenously on G15 with 101 micrograms [14C]FB1 (3.179 x 10(5) dpm). After 1 hr, which allowed for ca. 98% of the dose to be cleared from the maternal blood, negligible amounts of radioactivity were found in the fetuses. Together, these results indicate that the CM, and by inference FB1, did not have significant reproductive effects at doses which are minimally toxic, and further suggest that little in utero FB1 exposure occurred through G15.

2-Methoxyethanol (ME) produces testicular lesions characterized by pachytene spermatocyte degeneration in rats and guinea pigs which differ in onset, severity, and morphological characteristics. In the rat, degenerating spermatocytes appear necrotic at 24 hr, while in the guinea pig they appear apoptotic 96 hr after the start of three daily doses. To further examine if the spermatocyte degeneration in both species represented necrosis or apoptosis, the extent and nature of nuclear DNA fragmentation after ME exposure were assessed both visually using an in situ nucleotide 3' end-labeling (ISEL) procedure and by DNA gel electrophoresis. Testes from rats given a single oral dose of ME (200 mg/kg) showed the expected pachytene spermatocyte degeneration 24 hr after dosing, with the nuclear chromatin degradation typical of necrosis. In contrast, testes from guinea pigs given daily oral doses of ME (200 mg/kg) showed spermatocyte degeneration at only 96 hr after the start of dosing, with marked peripheral nuclear chromatin condensation characteristic of apoptosis. Coincident with the appearance of morphologic changes, degenerating spermatocytes in both species contained fragmented DNA as revealed by the ISEL procedure. The pattern of DNA fragmentation on agarose gels in both species consisted of ordered multiples or "ladders" of approximately 200 base pairs, a hallmark of apoptosis, with their appearance coincident with the time course of morphologic spermatocyte degeneration and ISEL staining. Preliminary data reveal the appearance of divalent metal cation-dependent endonuclease activity at pH 7.0 in ME-treated immature (24-day-old) rat testis that produces a similar pattern of DNA fragmentation and which appears to be distinct from activity associated with the spontaneous germ cell degeneration observed in testes of this age. In summary, in vivo ME exposure induces spermatocyte apoptosis in both the rat and guinea pig despite differing morphological classifications and time of onset of cell death. Future studies will focus on further characterization of the testicular endonuclease in the rat and the potential role of increased intracellular Ca2+ as a "triggering" stimulus in ME-induced spermatocyte apoptosis.

Phenolphthalein is a cathartic agent that is widely used in over-the-counter laxatives. Thirteen-week toxicity studies of phenolphthalein were performed using F344N rats and B6C3F1 mice. Rats and mice were fed ad libitum with a NIH 07 diet containing 0; 3000; 6000; 12,000; 25,000; or 50,000 ppm phenolphthalein. On a milligram per kilogram body weight basis, rats and mice fed 50,000 ppm phenolphthalein ingested more drug than would be expected during human laxative abuse. Phenolphthalein produced little evidence of toxicity in rats. There was slingtly lower weight gain among the 25,000 and 50,000 ppm groups. Treated rats showed elevated relative kidney weights (males only) and elevated absolute and relative liver weights at 12,000–50,000 ppm phenolphthalein. Rat serum bile acids were depressed early (Days 5 and 6) by phenolphthalein treatment. Several treatment-related toxic effects, however, were identified in mice who received more phenolphthalein per unit body weight than rats. Although there were no effects on body weight gain, elevated liver weights were noted in female mice receiving 6000–50,000 ppm phenolphthalein. The primary treatment-related findings that occurred during the mouse studies involved the reproductive and hematopoietic systems. Reproductive changes including depressed testis and right epididymal weights and sperm density, an elevated production of abnormal sperm, and morphologic alterations in seminiferous tubules occurred at all levels of exposure (3000–50,000 ppm). Hematopoietic changes included bone marrow hypoplasia (12,000–50,000 ppm), increased splenic hematopoiesis (males only; 25,000 and 50,000 ppm), and an elevated incidence of micronucleated erythrocytes (6000–50,000 ppm).

This review will expand on the themes presented by Heindel and Treinen (1988). As a prelude to describing where selected compounds act on the endocrine regulation of the testis and the theories about their mechanisms, we will briefly review some of the central pathways that underlie this control. After reviewing some studies that define the site of action of lead on the reproductive system, we will discuss the “signature” lesion caused by androgen deficiency, and then move on to an evaluation of the effects of an antiandrogen (flutamide) on the male reproductive system. Finally, some consideration will be given to alterations in hepatic function which modify circulating levels of androgens.

Routine processing of testicular tissue through 10% neutral buffered formalin (NBF) into paraffin produces severe cellular shrinkage which obscures most of the morphologic detail. The following studies were performed to compare different combinations of immersion fixatives and embedding media for optimal cellular detail in the final histologic sections. We examined sections of testes from rats, mice, and rabbits fixed in either NBF, Bouin's, Zenker's, or Helly's fixatives, and embedded in either standard paraffin or glycol methacrylate. The results were similar in all 3 species. In paraffin, Bouin's or Helly's fixatives produced the fewest artifacts, while in glycol methacrylate, NBF-fixed tissue showed the greatest intracellular detail and preservation. The merits and limitations of each method are discussed for the rat, with exceptions for the other species noted where appropriate. The use of glycol methacrylate as the support medium for sectioning makes high-quality tissue sections available from formalin-fixed testes.