A lack of contact of sperm with accessory sex gland secretions deregulates DNA methylation and imprinted gene expression in rodent embryos.

Increased oxidative DNA damage is observed in sperm devoid of contact with accessory sex gland (ASG) secretion. After fertilization, these sperm may produce abnormal embryos. In this study, we investigated the possibility that the pattern of DNA methylation and imprinted gene expression in these embryos may be perturbed. Epididymal sperm, uterine sperm, and embryonic day 13 (E13) embryos were collected from hamster and mouse. The extent of global DNA methylation was determined with an antibody against methylcytosine using an embryo DNA dot. The sperm and embryo Gtl2 promoter and H19 differential methylated region (DMR) were subject to bisulfite sequencing. Expression of their reciprocally activated genes Dlk1 and Igf2 was quantified by real-time PCR. Genome-wide DNA hypo-methylation in both hamster and mouse embryos sired by males without ASG was observed. The imprinting pattern of fetal mouse Gtl2 promoter and fetal hamster H19 DMR were also disrupted while the expression of Dlk1 and Igf2 was dysregulated in the hamster embryo. This study suggests that a lack of contact of sperm with the ASG secretion disrupts genome-wide DNA methylation and also affects the DNA methylation pattern of imprinted genes in embryos.

Absence of paternal accessory sex gland secretions disturbs epigenetic reprogramming and expression of Igf2 and Dlk1 in golden hamster embryos.

Accessory sex gland (ASG) secretion is known to exert an effect on sperm that is heritable in hamster embryos. We hypothesized that ASG secretion changes the sperm epigenome, which in turn is propagated in sired embryos. To test our hypothesis, we produced male hamsters that were devoid of either all ASG (TX) or only the ventral lobe of the prostate gland (VPX). A sham-operated control group (SH) was also established. These males were mated with normal females; uterine sperm, fertilized oocytes, and pre-implantation embryos were harvested from the females after mating. Epididymal sperm were collected at the end of experiments. Immunofluorescent staining was performed on these harvested specimens using antibodies against 5-methylcytosine, Dnmt1, Dnmt3a, Dnmt3b, protamine 1, protamine 2, and aectyl-H4K5. Expression of Igf2 and Dlk1 were analyzed by real-time RT PCR and in situ hybridization. We demonstrated that the DNA methylation pattern changed dynamically in SH, TX, and VPX fertilized oocytes. In VPX and TX embryos, DNA demethylation was slower and remethylation was delayed when compared with SH embryos. In addition, Dnmt3b expression was also abnormal. When sperm from VPX and TX males were exposed to whole ASG secretion in vivo, the resulting embryos all methylated normally. Immunofluorescent staining revealed that there was no difference in protamine packaging of uterine sperm from VPX and TX males. The staining also showed a lower level of acetyl-H4K5 expression in the male pronuclei of TX produced embryos. Furthermore, the VPX and TX embryos also expressed higher levels Igf2, and Dlk1. We concluded that interactions between ASG and sperm affected: (1) histone acetylation in male pronuclei; (2) DNA methylation in fertilized oocytes; and (3) Igf2 and Dlk1 expression embryos.

Ablation of paternal accessory sex glands imparts physical and behavioural abnormalities to the progeny: an in vivo study in the golden hamster.

The functional significance of male accessory sex glands (ASG) remains unclear. This study explored their importance in reproduction. In previous investigations, embryos sired by males with ASG either totally or partially removed had a shift in the cell cycle and delayed cleavage during preimplantation development, higher incidence of apoptosis, early oviductal-uterine transit, higher proportion of embryo degeneration, lower implantation rate, and ultimately reduced fertility and fecundity. Some pups were born alive; but would they be normal? We hypothesized that the first generation offspring (F1) could also bear undesirable traits. To test our hypothesis, we raised and studied these F1 pups from birth to 8 weeks. We monitored physical growth and assessed behaviour such as nest patch odor preference, acoustic startle response (ASR) and exploratory activity. We detected deviations from the norm in physical growth, a premature cessation of nest patch odor preferences, accelerated acoustic startle habituation and more frequent rearing when exposed to a novel environment. In terms of structure, we found one incidence of diphallus with duplicated urethra. We concluded that sperm lacking contact with ASG secretions gave rise to progeny with abnormal traits.

Male genital tract antioxidant enzymes--their ability to preserve sperm DNA integrity.

Male germ cells are unique because they lose a bulk of their cytoplasm as cytoplasmic droplets when they develop, leading to a decrease in endogenous antioxidant and hence a dependence on extracellular antioxidant system to overcome oxidative stress. Spermatozoa are particularly vulnerable to oxidative stress because their plasma membrane is rich in polyunsaturated fatty acids and membrane-bound NADPH oxidase. To protect spermatozoa from oxidative attack, an optimal amount of reactive oxygen species is maintained by balancing the reactive oxygen species generated during sperm maturation in the epididymidis and antioxidants in secretions of the male reproductive tract. The male accessory sex glands secretions have been shown to be the major source of antioxidant enzymes in the ejaculate and have the important function of preserving sperm DNA integrity from oxidative stress experienced in the uterine environment. In our in vivo golden hamster model, ablation of the five major male accessory sex glands, namely the ampullary glands, coagulating glands, dorsolateral prostate, ventral prostate and seminal vesicle, was found to cause higher incidence and greater degree of DNA damage in spermatozoa. These damaged sperm are able to undergo fertilization at the same rate as intact ones; however, the outcome of embryos sired is seriously affected.

Comparative proteomic analysis identifies protein disulfide isomerase and peroxiredoxin 1 as new players involved in embryonic interdigital cell death.

In this study, we used comparative proteomics to identify proteins that were involved in the regulation of interdigital cell death. The protein profiles of embryonic day (E) 12.5 and 13.5 mouse hindlimb interdigital tissues were compared to identify proteins that were differentially expressed. The interdigital cells are irreversibly committed to programmed cell death (PCD) at E13.5, whereas they are developmentally plastic at E12.5. We established that protein disulfide isomerase (PDI) expression was up-regulated at E13.5, while peroxiredoxin 1 (Prdx1) expression was down-regulated at this time point. Semiquantitative reverse transcriptase-polymerase chain reaction and Western blot analyses confirmed the data obtained from the two-dimensional electrophoresis gels. Furthermore, we were able to up-regulate PDI expression by manipulating the E12.5 interdigital tissues to die during culture, although this up-regulation was not possible when cell survival was promoted. In addition, we could inhibit interdigital cell death and expression of proapoptotic genes (Bmp-4 and Bambi) by treating interdigital tissues with PDI antibodies and bacitracin (a PDI enzyme inhibitor). These findings suggested that PDI was involved in the activation and maintenance of interdigital cell death. Conversely, we determined that Prdx1 expression was maintained when interdigital cultures were manipulated to survive but down-regulated when the cultures were permitted to die. The result suggested that Prdx1 was involved in maintaining interdigital cell survival. However, we were unable to induce interdigital cell death by means of RNA interference-mediated silencing of Prdx1 expression, indicating that Prdx1 down-regulation is not sufficient for PCD to occur. Proteomic analysis of the Prdx1 knock-down cells revealed that the level of NF-kappaB inhibitor epsilon (IkappaBepsilon) was dramatically reduced. Furthermore, we found an increase in NFkappaB activation and reactive oxygen species (ROS) levels in the cytoplasm as a result of Prdx1 knockdown. We also found that silencing Prdx1 made the interdigital cells more susceptible to ROS-induced cell death. Taken together, our study identifies two new players in interdigital cell death and highlights that PCD is regulated by a delicate balance of proapoptotic and survival-promoting activities.

Tissue specific expression and sequence analysis of a stress responsive gene Bre in adult golden hamster (Mesocricetus auratus).

Bre (brain and reproductive organ-expressed) is a new and putative stress-modulating gene of yet unknown function. BRE has previously been shown to interact with type 1 tumor necrosis factor receptor (TNFR1) and modulate the action of TNF. Apart from the brain and reproductive organs, Bre and BRE are highly expressed in steroid producing tissues such as the adrenal gland. Here we report for the first time the cloning of the Bre gene from golden hamster, a model organism extremely valuable for reproduction and steroid research, and examination of its tissue specific expression. Sequence analysis demonstrated that the peptide sequence of BRE in hamster shares approximately 99% homology with those of human, monkey and mouse. The hamster Bre gene transcribed an approximately 1.8-kb mRNA which translated a 44-kDa protein. Bre was strongly expressed in neurons and luminal epithelia of urogenital, digestive and respiratory organs. Bre was also detected in lymphoid tissues and endocrine glands. Immunohistochemistry demonstrated a similar protein expression pattern. Exceptions to this included the adrenal gland, where a high level of Bre was accompanied by weak immunoreactivity; as well as the oocytes and islets of Langerhans, where BRE protein but not the mRNA was localized. These data indicated that Bre gene products were expressed in a wide variety of tissues other than the brain and reproductive organs, as was originally described. Based on our findings, we propose that Bre is a housekeeping gene in tissues that are constantly subjected to environmental hazards such as luminal epithelia. Our results further support the proposed role for BRE in endocrine and immune functions.

Heart-type fatty acid binding proteins are upregulated during terminal differentiation of mouse cardiomyocytes, as revealed by proteomic analysis.

At birth, the cardiomyocytes in the mouse neonatal heart still retain their ability to proliferate. However, this lasts only a few days and then the cardiomyocytes irreversibly lose their potential to divide. It is still not fully understood what factors are involved in the cessation of cardiomyocyte proliferation. Using proliferating cell nuclear antigen (PCNA) antibodies, we established that cardiomyocytes could divide extensively in 2-day-old mouse neonatal hearts and to a lesser extent in 6-day-old hearts. By 13 days, the cardiomyocytes have mostly stopped dividing. Comparative two-dimensional gel electrophoresis (2-DE) was performed on total proteins extracted from the 2-day- and 13-day-old hearts, in order to identify peptides that might be involved in the inhibition of cardiomyocyte proliferation. Using matrix-assisted laser desorption ionization mass spectroscopy (MALDI-TOF), we identified two protein spots that have the same molecular weight (approximately 14 kDa) but different pIs (5.9 and 6.1). Mass spectra analysis determined the proteins to be isoforms of the heart-type fatty acid binding protein (H-FABP). The pI 6.1 H-FABP is also known as mammary-derived growth inhibitor (MDGI; Specht et al. 1996). MGDI is a breast tumour growth suppressor gene capable of inhibiting tumour cell proliferation (Huynh et al. 1995). Both H-FABP isoforms were expressed in 2-day-old hearts but became strongly upregulated in 13-day-old hearts. We examined whether H-FABPs and PCNA were coexpressed in 2-, 6- and 13-day-old heart histological sections, using MDGI antibodies. The antibody could detect both forms of H-FABPs. It was established that there was a correlation between an increase in H-FABP expression and a decrease in PCNA expression. Hence, we tentatively propose that H-FABP isoforms are involved in regulating cardiomyocyte growth and differentiation in mouse neonatal hearts.

Growth arrest-specific 2 gene expression during patellar tendon healing.

We examined the cellular and molecular processes involved in patellar tendon healing following induced injury. A wound was surgically created at the center of the patellar tendon of adult rats. The wound site was examined at selected time intervals by immunohistochemical and in situ hybridization techniques. It was found that, between the 2nd and 7th day postoperation, fibroblast-like cells invaded the wound site. DiI-labelling experiments suggested that the majority of cells that occupied the wound originated from the edges of the wound. Furthermore, immunohistochemical studies revealed that at the wound site a meshwork of fibronectin developed that can support the migration of the DiI-labelled cells. We also examined the spatial and temporal expression patterns of the growth arrest specific 2 (GAS2) gene during patellar tendon healing. GAS2 was found strongly expressed in the tenocytes of unoperated patellar tendons. The gene was also expressed in the intact regions of operated tendons but not in the fibroblast-like cells that occupied the wound site, when examined 2 days postoperation. In addition the strip of intact tendon directly opposite the wound site also did not express GAS2. Examination of the experimental tendon at the 3rd month, when cells had completely occupied the wound site, revealed that Gas2 was expressed by all cells found in the wound. Bromodeoxyuridine (BrdU) incorporation analysis revealed that the presence of Brdu-positive cells in the wound indirectly correlated with the absence of Gas2 expression. We speculate that the GAS2 gene might play a role in regulating tenocyte proliferation during tendon healing.

Embryos sired by males without accessory sex glands induce failure of uterine support: a study of VEGF, MMP and TGF expression in the golden hamster.

To account for reproductive failure induced by surgical deletion of paternal accessory sex glands in the golden hamster in vivo, we studied expression of vegf, FLT-1 (VEGF-R1), FLK-1 (VEGF-R2), MMP and TGF-beta in endometrium of the dam and sired embryos during 5-7 days post coitum by immunohistochemistry, in situ hybridisation, semiquantitative RT-PCR and enzyme-linked immunosorbent assay. Spatiotemporal pattern of vegf expression in the control animals was similar to that reported for intact animals by our group. Removal of paternal ampullary glands did not disturb the normal expression pattern. Removal of ventral prostate glands alone or all accessory sex glands was associated with reduction of vegf transcripts and protein levels in both the embryo and endometrium. FLT-1, FLK-1 and MMP-2 were also reduced. MMP-1 was not changed whereas TGF-beta1 expression was enhanced. There was no expression in endometrium in between implantation sites. Thus the implanted embryos had a trophic effect on growth factor production by the endometrium, and the levels of expression were determined by viability and structural integrity of the conceptus. Based on these findings we concluded that incompetent embryos sired by males without the ventral prostate gland or all accessory sex glands reduced the potential of the uterus to support pregnancy. A negative cycle of events was thus set up and eventually led to premature termination of pregnancies.

Protection of sperm DNA against oxidative stress in vivo by accessory sex gland secretions in male hamsters.

Reactive oxygen species scavengers present in male accessory sex gland secretions might afford antioxidant protection to sperm DNA. This study was conducted to determine whether accessory sex gland secretions protect the genome and function of spermatozoa against oxidative damage in the uterus. Male golden hamsters were divided into four experimental groups: (i) all accessory sex glands removed; (ii) ampullary glands removed; (iii) ventral prostate gland removed and (iv) sham-operated controls. Ejaculated spermatozoa recovered from uteri 15-30 min after mating with experimental males and caput and cauda epididymal spermatozoa obtained from intact males were incubated in 0-20 mmol NADPH l(-1) for 2 h. These spermatozoa and untreated uterine spermatozoa were processed for two types of comet assay (single cell gel electrophoresis): alkaline comet assay (pH > 13) which revealed single-strand DNA breakage and neutral comet assay (pH 9) which revealed double-strand DNA breakage. In comparison with the sham-operated controls, spermatozoa that had not been exposed to accessory sex gland secretions had a higher incidence and more extensive single-strand DNA damage with increasing concentrations of NADPH. Spermatozoa from hamsters without ampullary glands and from hamsters without the ventral prostate glands were similar to those of the control group. After incubation with NADPH, the capacity of spermatozoa from hamsters without accessory glands and from sham-operated controls to fuse with oocytes in vitro was reduced. However, only hamsters without accessory glands showed a negative correlation between single-strand DNA damage and sperm-oocyte fusion. Cauda epididymal spermatozoa were less susceptible to NADPH treatment compared with caput epididymal spermatozoa. The results of the present study showed that male accessory sex gland secretions can preserve the integrity of the sperm genome.

Total ablation of paternal accessory sex glands curtails developmental potential in preimplantation embryos in the golden hamster.

The effects of total removal of paternal accessory sex glands (TX) on preimplantation embryonic development was studied in the golden hamster model. Cell numbers of the two groups of embryos did not differ up to 60 h p.c., but at 66 and 70 h p.c., each TX embryo has 2 and 3 cells less respectively (P<0.05, TX vs SH). At 70 h p.c., 46.6+/-4.4 of the TX embryos blastomeres were labelled with the terminal deoxynucleotide transferase - mediated dUTP-nickend-labelling technique, compared with 31.5+/-2.1 in the SH group (P<0.01, TX vs SH). No difference was found in the SDS-PAGE profiles of two-cell embryos from the two groups. An extra band corresponding to 136.5 kDa was consistently found in the four-cell TX embryos. The nascent proteins profiles of four-cell embryos from the two groups were similar. As the embryos progressed from two to four cells, the protein content decreased by 16% in the SH embryos (P<0.05) and 7% in the TX embryos. These observations suggest that total ablation of paternal accessory sex glands could result in developmental aberrations from the two-cell to morula stages and a higher incidence of apoptosis at 70 h p.c.

Ablation of paternal accessory sex glands is detrimental to embryo development during implantation.

The accessory sex glands are present in most mammals, but their function(s) have not yet been clearly defined. In the golden hamster, removal of all the glands or the ventral prostate alone have been shown to considerably reduce fertility, while the effect is milder if the ampullary glands only are removed. In this study, embryo development from the 5th to the 7th day after mating are examined. Structural and morphometric criteria such as cell number, cell density, embryo volume, volume fraction of proamniotic cavity further revealed that abnormalities can be demonstrated as early as day 5 in the embryos sired by males with the ventral prostate gland alone or all glands ablated. Twin implantation and deviation from normal implanted axis are also observed. This is likely to be attributed to attenuated cell proliferation, as indicated by proliferating cell antigen labelling and more necrotic cell death. Taken together, exposure of sperm to secretions of the male accessory sex glands in particular, the ventral prostate, is important for differentiation and multiplication of cells after the embryo has implanted.

The growth arrest specific gene (gas6) protein is expressed in abnormal embryos sired by male golden hamsters with accessory sex glands removed.

Expression of growth arrest specific gene (gas6) and its receptors in embryonic and uterine tissues in normal pregnancy and pregnancy that produces abnormal embryos sired by hamsters with partial or total deletion of male accessory sex glands was studied by in situ hybridization, immunohistochemistry, reverse-transcription polymerization reaction and enzyme-linked immunoabsorbant assay. At oestrus, very strong gas6 mRNA and Gas6 expression were seen only in the uterine epithelium and endometrial glands. Upon implantation, both of them could be demonstrated in the decidualizing stroma. From day 4 to day 7 p.c, gas6 mRNA was present in the embryo, but Gas6 immunoreactivity was only found in those showing features of degeneration. The gas6:beta-actin mRNA ratio was low in oestrus and at day 4 of pregnancy but rose as the embryo grew. As for the receptors, Rse was detected in embryonic cells during days 5-7 p.c., and decidual cell from days 4 to 7 p.c., but Mer could be found in decidual cells and trophoblasts. It was concluded that gas6 had a role in endometrial transformation during decidualization and trophoblastic invasion. In the embryo, gas6 was transcribed, but the protein was only produced in response to need, such as when normal progression of development was threatened.

Functions of the growth arrest specific 1 gene in the development of the mouse embryo.

The growth arrest specific 1 (gas1) gene is highly expressed in quiescent mammalian cells (Schneider et al., 1988, Cell 54, 787-793). Overexpression of gas1 in normal and some cancer cell lines could inhibit G(0)/G(1) transition. Presently, we have examined the functions of this gene in the developing mouse embryo. The spatial-temporal expression patterns for gas1 were established in 8.5- to 14.5-day-old embryos by immunohistochemical staining and in situ hybridization. Gas1 was found heterogeneously expressed in most organ systems including the brain, heart, kidney, limb, lung, and gonad. The antiproliferative effects of gas1 on 10.5 and 12.5 day limb cells were investigated by flow cytometry. In 10.5 day limbs cells, gas1 overexpression could not prevent G(0)/G(1) progression. It was determined that gas1 could only induce growth arrest if p53 was also coexpressed. In contrast, gas1 overexpression alone was able to induce growth arrest in 12.5 day limb cells. We also examined the cell cycle profile of gas1-expressing and nonexpressing cells by immunochemistry and flow cytometry. For 10.5 day Gas1-expressing heart and limb cells, we did not find these cells preferentially distributed at G0/G1, as compared with Gas1-negative cells. However, in the 12.5 day heart and limb, we did find significantly more Gas1-expressing cells distributed at G0/G1 phase than Gas1-negative cells. These results implied that Gas1 alone, during the early stages of development, could not inhibit cell growth. This inhibition was only established when the embryo grew older. We have overexpressed gas1 in subconfluent embryonic limb cells to determine the ability of gas1 to cross-talk with various response elements of important transduction pathways. Specifically, we have examined the interaction of gas1 with Ap-1, NFkappaB, and c-myc responsive elements tagged with a SEAP reporter. In 10.5 day limb cells, gas1 overexpression had little effect on Ap-1, NFkappaB, and c-myc activities. In contrast, gas1 overexpression in 12.5 day limb cells enhanced AP-1 response while it inhibited NFkappaB and c-myc activities. These responses were directly associated with the ability of gas1 to induce growth arrest in embryonic limb cells. In the 12.5 day hindlimb, gas1 was found strongly expressed in the interdigital tissues. We overexpressed gas1 in these tissues and discovered that it promoted interdigital cell death. Our in situ hybridization studies of limb sections and micromass cultures revealed that, during the early stages of chondrogenesis, only cells surrounding the chondrogenic condensations expressed gas1. The gene was only expressed by chondrocytes after the cartilage started to differentiate. To understand the function of gas1 in chondrogenesis, we overexpressed the gene in limb micromass cultures. It was found that cells overexpressing gas1/GFP could not participate in cartilage formation, unlike cells that just express the GFP reporter. We speculated that the reason gas1 was expressed outside the chondrogenic nodules was to restrict cells from being recruited into the nodules and thereby defining the boundary between chondrogenic and nonchondrogenic forming regions.

Bmp-4 requires the presence of the digits to initiate programmed cell death in limb interdigital tissues.

The effects of Bmp-4 on interdigital cell death were investigated in the mouse. Affi-Gel beads, loaded with recombinant Bmp-4 protein, were transplanted into the interdigital tissues of day 12.5 hindlimb, ex utero. It was established that Bmp-4 could induce precocious interdigital cell death. Using in situ hybridization, the expression patterns of bmp-4 and alk-6 receptor were established. Both genes were found coexpressed in the interdigital region of 12.5- and 13. 5-day hindlimbs. This suggests that Bmp-4 may act in an autocrine fashion. We have also studied the effects of Bmp-4 on 12.5-day interdigital tissue cultures. In all specimens examined, the interdigital tissues produced cartilage instead of participating in cell death. The addition of exogenous Bmp-4 to the interdigital cultures did not induce apoptosis but instead enhanced chondrogenesis. The discrepancy between the effects of Bmp-4 in vitro and ex utero was attributed to the presence of digits. When a flanking digit was left attached to the interdigital tissues, in vitro, Bmp-4 promoted apoptosis instead of chondrogenesis. In sum, the results suggest that Bmp-4 is a multifunctional protein and its effect on the interdigital tissues is dependent on the modulating influence of the digits.

Expression of vascular endothelial growth factor (VEGF) and its receptors during embryonic implantation in the golden hamster (Mesocricetus auratus).

Expression of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) and its receptors (flt-1 and flk-1) during the peri-implantation period (days 3, 4, 5, 6 and 7 post coitus) in the golden hamster was investigated by in situ hybridization, immunohistochemistry and the reverse transcription/polymerase chain reaction (RT-PCR). Three days after mating, in situ hybridization and immunohistochemical staining revealed weak VEGF expression only in the uterine epithelium; this expression was similar to that seen at oestrus. Flt-1 but no flk-1 immunoreactivity was observed. At day 4, the subepithelial stroma and embryo displayed immunoreactivity for VEGF and flt-1, whereas endothelial cells expressed both flt-1 and flk-1. At day 5, immunoreactivity for both VEGF and its receptors was detected in decidual cells and vascular endothelial cells. Only a few embryonic cells expressed VEGF mRNA but strong signals were noted in decidual cells. The patterns of VEGF and VEGF receptor expression were the same in the day-6 and day-7 embryos and decidua, except for an increase in intensity as development progressed. Based on these findings, we conclude that, in addition to its known actions on endometrial angiogenesis and tissue swelling, VEGF may also facilitate the proliferation and differentiation of the endometrium and help to sustain the avascular embryo during this early stage of development.

Male accessory sex gland secretions affect oocyte Ca2+ oscillations during in-vitro fertilization in golden hamsters.

To evaluate the effect of male accessory sex gland secretions on Ca2+ oscillations of oocytes, epididymal or ejaculated spermatozoa recovered from uteri were used to inseminate oocytes. Ca2+ oscillations were measured by Fura 2 fluorescence imaging (F340/F380). We showed that although Ca2+ oscillations induced by ejaculated spermatozoa had a pattern similar to those induced by epididymal spermatozoa, the amplitude of the first Ca2+ transient in the former group was significantly higher (P < 0.05) and the duration was significantly longer (P < 0.01). Oocytes inseminated with ejaculated spermatozoa recovered from uteri from males had ampullary glands or ventral prostates removed showed significantly lower Ca2+ oscillations compared to the controls (P < 0.05, P < 0.01 respectively). Moreover, the relative area of the first Ca2+ transient in treatment groups was significantly smaller than the control. In addition, a significantly higher percentage of oocytes (52%) inseminated by spermatozoa from males with all accessory sex glands removed showed non-oscillatory Ca2+ transients, compared to the controls (5%, P < 0.05). These results indicate that accessory sex gland secretions can affect Ca2+ oscillations. The differences between Ca2+ oscillations induced by epididymal and uterine spermatozoa from males with all accessory sex glands removed suggest that uterine factors may also influence this process.

Effects of male accessory sex glands on sperm decondensation and oocyte activation during in vivo fertilization in golden hamsters.

Removal of paternal male accessory sex glands (ASG) could cause a delay in DNA synthesis in hamster zygotes fertilized in vivo. In view of the fact that this process is closely related to pronuclear development which, in part, depends on sperm nuclear decondensation and oocyte activation during fertilization, we carried out a series of experiments were undertaken to determine whether ASG also has an effect on these early events. (1) Oocytes were collected from females mated with SH (sham-operated control), AGX (bilateral excision of ampullary glands), VPX (bilateral excision of ventral prostates) or TX (excision of all ASG) males (n = 8 per group) at 4, 5 and 6 h post coitus. (2) Epididymal spermatozoa were incubated with total ventral prostate (VP) secretion to study its effect on dithiothreitol-induced sperm decondensation. (3) Histone H1 kinase activity in oocytes collected as described in (1) was determined. (4) Exocytosed cortical granules on oocytes were labelled with FITC-LCA and quantified by a Metamorph Imaging System. Results showed that sperm decondensation and resumption of meiosis in oocytes in VPX and TX groups were significantly slower compared with SH. VP secretion augmented sperm decondensation in vitro. At 4 h post coitus, the relative activity of histone H1 kinase in the TX and VPX groups was significantly higher than that in the SH group (p < 0.01). Cortical granule exocytosis in the AGX group was consistently weaker at all time points studied and was significantly lower than that of the control at 4 h post coitus (p < 0.05), while the percentage of polyspermic fertilization in the AGX group was significantly higher compared with that in the SH group (p < 0.05). Taken together, these results show that the lack of exposure of spermatozoa to secretions of the ASG does not jeopardize their ability to penetrate ova, although other aspects of their function in the early stages of gamete interaction and subsequent initiation of embryonic development are affected.

gas2 is a multifunctional gene involved in the regulation of apoptosis and chondrogenesis in the developing mouse limb.

The growth-arrest-specific 2 (gas2) gene was initially identified on account of its high level of expression in murine fibroblasts under growth arrest conditions, followed by downregulation upon reentry into the cell cycle (Schneider et al., Cell 54, 787-793, 1988). In this study, the expression patterns of the gas2 gene and the Gas2 peptide were established in the developing limbs of 11.5- to 14. 5-day mouse embryos. It was found that gas2 was expressed in the interdigital tissues, the chondrogenic regions, and the myogenic regions. Low-density limb culture and Brdu incorporation assays revealed that gas2 might play an important role in regulating chondrocyte proliferation and differentiation. Moreover, it might play a similar role during limb myogenesis. In addition to chondrogenesis and myogeneis, gas2 is involved in the execution of the apoptotic program in hindlimb interdigital tissues-by acting as a death substrate for caspase enzymes. TUNEL analysis demonstrated that the interdigital tissues underwent apoptosis between 13.5 and 15.5 days. Exactly at these time points, the C-terminal domain of the Gas2 peptide was cleaved as revealed by Western blot analysis. Moreover, pro-caspase-3 (an enzyme that can process Gas2) was cleaved into its active form in the interdigital tissues. The addition of zVAD-fmk, a caspase enzyme inhibitor, to 12.5-day-old hindlimbs maintained in organ culture revealed that the treatment inhibited interdigital cell death. This inhibition correlated with the absence of the Gas2 peptide and pro-caspase-3 cleavage. The data suggest that Gas2 might be involved in the execution of the apoptotic process.

Neuroendocrinology of melatonin in reproduction: recent developments.

The circadian melatonin rhythm with high levels in the dark period is important for the synchronization of reproductive response to appropriate environmental conditions in animals. The target sites of melatonin action on reproductive functions remain to be clarified. Using autoradiography (ARG) and radioreceptor binding assays with 2[125I]iodomelatonin, a melatonin agonist, as the radioligand, studies on the sites of melatonin action have increased significantly in the last ten years. The recent cloning of melatonin receptor subtypes also allowed the characterization of receptor(s) to the molecular level. Earlier reports have documented that the hypothalamic-pituitary axis plays a vital role in the regulation of reproduction by melatonin. This is supported in part by the demonstration of melatonin receptors in the suprachiasmatic nuclei (SCN) in the brain and pars tuberalis (PT) in the pituitary. However, the nature of SCN and PT involvement in the reproductive action of melatonin remains unknown. In addition to the hypothalamus and pituitary, the two classical sites of melatonin action, other targets have been identified. The recent demonstration of 2[125I]iodomelatonin binding sites or melatonin receptors in the testis, epididymis, vas deferens, prostate, ovary and mammary gland suggest the concept of multiple sites of melatonin action on the reproductive system. The presence of melatonin receptors in the said tissues is consistent with earlier reports of direct melatonin actions on different levels of the reproductive system. This multiple levels of melatonin action, from the hypothalamus, pituitary, gonads to other reproductive tissues form a robust system of photoperiodic control in animal reproduction. This would guarantee successful gestation and delivery of the offspring at a time with optimum food availability and ultimately favourable for the survival of species. Molecular and cellular studies of melatonin signaling system(s), its regulation and effects on downstream functional events in the future may provide new insights and directions for the study of the physiology and pharmacology of fertility and contraception in animals and humans.