Persistence of müllerian ducts in male rabbits passively immunized against bovine anti-müllerian hormone during fetal life

A female rabbit was immunized against purified bovine AMH and mated. Booster injections were given at Day 8 of pregnancy to ensure a high titer of anti-AMH antibodies at the time the rabbit fetal testis begins to produce AMH. In three consecutive litters, the immunized female produced a total of 12 males, 9 of which had persistent Müllerian duct derivatives. No other significant abnormalities were detected in these animals, which were compared to the offspring of a control saline-injected female. In particular, testicular morphology was normal in most animals, and serum FSH levels did not differ from controls. This experimental model lends no support to the hypothesis that AMH controls extra-Müllerian events of male sex differentiation, nor that of the existence of a regulatory mechanism for synthesis of AMH by Sertoli cells, but it does not definitely exclude these possibilities, inasmuch as our tentative conclusions are based upon study of only one immunized female.     

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

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

Do male roe deer clump together during the rut?

Movement s an d habitat use of 7 male and 7 female roe deerCapreolus capreolus (Linnaeus, 1758) were studied by radioteleme try from March 1999 to February 2001. Annual and bimonthly home ranges of males were small (ca 10 ha, 95% kernel), with large overlap among individuals throughout the year. Exclusive core areas (ca 0.4 ha, 25% kernel) were concentrated in the forest, a limited and sought-after resource in the study area. The difference in overlap between male exclusive core areas and female home ranges in the pre-rut and rut periods suggested that females made excursions to search for territorial males during the rut Our results support the mating strategy hypothesis of territorial behaviour Different space use patterns occurred between the sexes, with females apparently playing an active role in mate choice by visiting males at clumps of core areas in the forest.     

Nm23/NDP kinases in human male germ cells: role in spermiogenesis and sperm motility?

Nucleoside diphosphate (NDP) kinases, responsible for the synthesis of nucleoside triphosphates and produced by the nm23 genes, are involved in numerous regulatory processes associated with proliferation, development, and differentiation. Their possible role in providing the GTP/ATP required for sperm function is unknown. Testis biopsies and ejaculated sperm were examined by immunohistochemical and immunofluorescence microscopy using specific antibodies raised against Nm23-H5, specifically expressed in testis germinal cells and the ubiquitous NDP kinases A to D. Nm23-H5 was present in sperm extract, together with the ubiquitous A and B NDP kinases (but not the C and D isoforms) as shown by Western blotting. Nm23-H5 was located in the flagella of spermatids and spermatozoa, adjacent to the central pair and outer doublets of axonemal microtubules. High levels of NDP kinases A and B were observed at specific locations in postmeiotic germinal cells. NDP kinase A was transiently located in round spermatid nuclei and became asymmetrically distributed in the cytoplasm at the nuclear basal pole of elongating spermatids. The distribution of NDP kinase B was reminiscent of the microtubular structure of the manchette. In ejaculated spermatozoa, the proteins presented specific locations in the head and flagella. Nm23/NDP kinase isoforms may have specific functions in the phosphotransfer network involved in spermiogenesis and flagellar movement.     

Involvement of signaling of VEGF and TGF-β in differentiation of sinusoidal endothelial cells during culture of fetal rat liver cells

Embryonic development of the liver is closely associated with vascular organization. However, little is known about the mechanisms of vascular differentiation during liver development. Our previous study showed that the maturation of sinusoidal endothelial cells (SECs) occurred during embryonic day 13.0 (E13.0) to E15.0. To improve our understanding of SEC differentiation, we examined here the expression of maturation markers, SE-1 and stabilin-2, in fetal livers and also attempted to establish an in vitro SEC differentiation system by culturing E13.5 fetal liver cells. Immunohistochemical examination of SE-1 and stabilin-2 expression during fetal rat liver development revealed that these differentiation markers were co-expressed in SECs in the late stage of liver development, although stabilin-2 was expressed in almost all vascular endothelial cells in the early stage. Liver cells from the E13.5 rat fetus were cultured in EBM-2 medium containing vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGF-β1) and VEGF plus SB-431542 (an inhibitor of the TGF-β1 receptor, activin receptor-like kinase 5 [ALK-5]). In vitro SEC differentiation, as indicated by the appearance of cells co-expressing SE-1 and stabilin-2 and of cells with cytoplasmic fenestrae in endothelial sheets, was induced by the addition of both VEGF and SB-431542, an inhibitor of the phosphorylation of Smad2/3 but not that of Smad1/5/8 in the cultured cells. These results indicate for the first time that both VEGF signaling and the blocking of the ALK-5-Smad2/3 signal pathway are important for the fetal differentiation of SECs.     

Multipoint linkage map of the human pseudoautosomal region,based on single-sperm typing: do double crossovers occur during male meiosis?

Sperm typing was used to measure recombination fractions among pseudoautosomal markers and the beginning of the X/Y-specific sequences located at the pseudoautosomal boundary. These experiments included primer-extension preamplification and PCR followed by allele typing using gel electrophoresis. A newly developed data-analysis program allowed the construction of the first multipoint-linkage sperm-typing map, using results obtained on seven loci from three individuals. The large sample size not only confirmed the increased recombination activity of the pseudoautosomal region but allowed an estimate of interference of recombination to be made. The coefficient of coincidence was calculated to be .26 over a physical distance of only approximately 1,800 kb. The observation of a few sperm presumably resulting from double recombination argues that more than one crossover event can occur in this region during male meiosis.     

CAMP activates Rap1 in differentiating mouse male germ cells: a new signaling pathway mediated by the cAMP-activated exchange factor Epac?

Rap1, a Ras-like G-protein, is implicated in the signaling of various cellular processes as morphogenesis, differentiation, cell adhesion and spreading, and maintenance of T cell anergy and B cell activation. The effectors that mediate Rap1 signaling have not yet been definitely identified, with the exception of B-Raf which, however, is restricted to neuronal tissues and a small subset of other cell types, including in particular male germ cells. We previously showed that in mouse spermatids Rap1 could interact with B-Raf giving rise to a signaling complex. Here we investigated about the possible molecules which "switch on" Rap1 finding that cAMP could in vivo activate endogenous Rap1. Spermatid-enriched cell cultures stimulated with 8-(4-chlorophenylthio)-cyclic AMP yielded higher levels of GTP-bound Rap1 than unstimulated cells. Since cAMP-induced Rap1 activation is actually retained to occur through Epac, we checked whether this recently discovered Rap1 exchange factor is expressed in male germ cells. Our findings indicate that Epac is present in spermatogenic cells and exhibits a preferential subplasmalemmal localization, although it shows also an intracellular location, more or less pronounced depending on the type of spermatogenic cell examined. Taken together, our data show that cAMP activates Rap1 in differentiating male germ cells which express the cAMP sensor Epac, thus suggesting that this activation might occur directly through Epac.     

Immunohistochemical analyses of cell–cell interactions during hepatic organoid formation from fetal mouse liver cells cultured in vitro

Cell–cell interactions among cell types constituting the fetal liver such as hepatoblasts, stellate cells and endothelial cells lead to functional lobule development. The present study was undertaken to investigate hepatic histogenesis in the primary culture of E12.5 mouse livers, including cell–cell and cell–matrix interactions. Fetal livers were dispersed with protease treatment and cultured for 5 days. Cellular adhesion of each hepatic cell type, gene expression and extracellular matrix deposition were analyzed by immunohistochemistry and immunoblotting. Immunohistochemical analysis demonstrated that the primary culture of fetal liver cells contained at least hepatoblasts, mesenchymal cells, endothelial cells, hemopoietic cells and Kupffer cells. Although hepatoblasts, mesenchymal cells, and endothelial cells aggregated separately in the initial step, they then formed a spheroid together, adhering to the glass slide, which led to the formation of flattened hepatic organoids. Hepatoblasts more preferentially adhered to mesenchymal cells than endothelial cells. Several extracellular matrix depositions were seen in aggregates consisting of at least hepatoblasts and mesenchymal cells within 12 h, but were poor in those lacking hepatoblasts. These data show that the primary culture of fetal liver cells contains most cell types constituting fetal livers, and may be useful for studying cell–cell interactions during liver development.     

IL-10 secreting CD21+ B cells are present in jejunal Peyer’s patches of sheep during fetal development

We recently reported a novel interleukin-10 (IL-10)-secreting CD21⁺ B cell population in jejunal Peyer’s patches (JPP) of sheep with a regulatory function (B_regs) suppressing Toll-like receptor 9 (TLR9)-induced cytokine responses. However, little is known about the development of these cells. Therefore, we investigate their existence in JPP cells from fetal and newborn lambs. CD21⁺ B cells were purified from JPP cells by magnetic cell sorting and subsequently stimulated with the TLR9 agonist, CpG ODN (CpG oligodeoxynucleotide). Lymphocyte proliferative responses, cytokine production (IL-10, IL-12 and interferon-γ [INF-γ]) and antibody secretion were assayed. We found that fetal and neonatal CD21⁺ B cells spontaneously secreted high levels of IL-10 regardless of CpG stimulation but that these cells did not produce any IL-12 or INF-γ upon stimulation with CpG. The observed responses are consistent with those previously reported for B_regs characterized in JPP of older lambs. Surprisingly, unlike in older lambs, fetal and neonatal JPP CD21⁺ B cells proliferated in response to CpG stimulation. Our investigations of fetal and neonatal lambs provide evidence for the development of IL-10-secreting CD21⁺ B cells in PPs prior to antigen exposure.     

Do competing males cooperate? Familiarity and its effect on cooperation during predator inspection in male three‐spined sticklebacks (Gasterosteus aculeatus)

We investigated the trade‐off between conflict and cooperation, using predator inspection behaviour in sticklebacks as a model system. Male three‐spined sticklebacks ( Gasterosteus aculeatus ) compete for territories during the breeding season and it has been demonstrated that the level of aggression between territorial neighbours declines with time, a phenomenon known as the dear enemy effect. In this experiment we examine whether this increase in familiarity between territorial neighbours can facilitate an increase in cooperation during predator inspection events. This was analyzed using male sticklebacks from four pond populations, two with and two without predatory rainbow trout ( Oncorhyncus mykiss ). Males were each exposed to five different treatments: (1) the presentation of a live rainbow trout when alone; (2) with a newly revealed territorial neighbour; (3) with this neighbour after two days of familiarization; (4) with another unfamiliar neighbour (termed a 'floater'); and (5) a second solitary trial (to provide controls at the start and end of the experiment). As predicted, fish from predator‐sympatric populations showed higher levels of predator inspection and lower rates of misdirected territorial aggression towards the predator throughout. However, familiarity between neighbouring males did not facilitate an increase in predator inspection behaviour. Instead, predator inspection behaviour decreased throughout treatments involving the presence of any sort of neighbouring male. Familiarity between neighbours did not influence their ability to cooperatively inspect, but only the nature of any aggressive territorial behaviours, all of which detracted equally from individual inspection effort.     

Development of the ovary and ontongeny of mRNA and protein for P450 aromatase (arom) and estrogen receptors (ER) α and β during early fetal life in cattle

Estradiol-17β is the predominant steroid produced during early stages of ovarian development in ruminants and steroid hormones have been hypothesized to regulate ovigerous cord formation, germ cell meiosis and ovarian vascular development. Therefore, the objective was to determine the presence and localization of mRNA and protein encoding cytochrome P450 aromatase (P450arom), and estrogen receptors α (ERα) and β (ERβ) during ovarian development in fetuses of cattle on days 35, 45, 60, 75, 90 and 105 after breeding (n = 4/age) using in situ hybridization and immunohistochemistry. No ovarian tissue was found in the day 35 fetuses, but was found in all later ages studied. There appeared to be little organization of specific structures in ovaries on days 45 and 60, although germ cells could be identified. Evidence of the beginning of ovigerous cord formation was found on day 60. By day 75 of gestation, the ovigerous cords were more extensive and mesonephric-derived cell streams were detectable. By day 90 (and still present at day 105), both ovigerous cords and cell streams/rete tubules were definitive structures of the developing ovaries. Ovaries appeared to develop in “lobular” segments around the periphery of the ovary. Some lobes appeared to be at slightly different developmental stages, as assessed by the extent or definition of ovigerous cord formation.The localization of mRNAs for P450arom, ERα and ERβ were closely associated with protein content. At days 45 and 60, mRNA and protein of P450arom and ERβ were located throughout ovaries with signal in medulla being denser than in the cortex. P450arom mRNA or protein was punctate, but not evident in germ cells. From day 75, P450arom was increasingly becoming localized to cell streams or clusters of cells (rete tubules) in the medulla, and by days 90 and 105 of gestation, was more definitively localized to cell streams and/or rete tubules. Similar to P450arom, ERβ mRNA and protein were observed in cells in the medulla, and also in germ cells, pre-granulosa cells and some surface epithelial cells. ERα mRNA and protein were predominately in the surface epithelium in ovaries of all ages with fainter signal for ERα protein also being observed in pre-granulosa and stromal cells including the cell streams/rete tubules. ERα protein was also detected in a few germ cells at days 90 and 105 of gestation. Thus, in cattle, estradiol-17β has the potential to regulate, in an autocrine/paracrine manner, a number of different cell types during ovarian development.     

Exposure to a maternal n-3 fatty acid-deficient diet during brain development provokes excessive hypothalamic–pituitary–adrenal axis responses to stress and behavioral indices of depression and anxiety in male rat offspring later in life

Brain docosahexaenoic acid (DHA, 22:6n-3) accumulates rapidly during brain development and is essential for normal neurological function. The aim of this study was to evaluate whether brain development was the critical period in which DHA deficiency leads to dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis in response to stress later in life. Rats were exposed to an n-3 fatty acid-deficient diet or the same diet supplemented with fish oil as an n-3 fatty acid-adequate diet either throughout the preweaning period from embryo to weaning at 3 weeks old or during the postweaning period from 3 to 10 weeks old. Exposure to the n-3 fatty acid-deficient diet during the preweaning period resulted, at weaning, in a significant decrease in hypothalamic DHA levels and a reduced male offspring body weight. DHA deficiency during the preweaning period significantly increased and prolonged restraint stress-induced changes in colonic temperature and serum corticosterone levels, caused a significant increase in GABA_A antagonist-induced heart rate changes and enhanced depressive-like behavior in the forced swimming test and anxiety-like behavior in the plus-maze test in later life. These effects were not seen in male rats fed the n-3 fatty acid-deficient diet during the postweaning period. These results suggest that brain development is the critical period in which DHA deficiency leads to excessive HPA responses to stress and elevated behavioral indices of depression and anxiety in adulthood. We propose that these effects of hypothalamic DHA deficiency during brain development may involve a GABA_A receptor-mediated mechanism.