Mammalian fertilization is dependent upon a series of bicarbonate-induced, cAMP-dependent processes sperm undergo as they "capacitate," i.e., acquire the ability to fertilize eggs. Male mice lacking the bicarbonate- and calcium-responsive soluble adenylyl cyclase (sAC), the predominant source of cAMP in male germ cells, are infertile, as the sperm are immotile. Membrane-permeable cAMP analogs are reported to rescue the motility defect, but we now show that these "rescued" null sperm were not hyperactive, displayed flagellar angulation, and remained unable to fertilize eggs in vitro. These deficits uncover a requirement for sAC during spermatogenesis and/or epididymal maturation and reveal limitations inherent in studying sAC function using knockout mice. To circumvent this restriction, we identified a specific sAC inhibitor that allowed temporal control over sAC activity. This inhibitor revealed that capacitation is defined by separable events: induction of protein tyrosine phosphorylation and motility are sAC dependent while acrosomal exocytosis is not dependent on sAC.

Although it is well established that the circadian clock regulates mammalian reproductive physiology, the molecular mechanisms by which this regulation occurs are not clear. The authors investigated the reproductive capacity of mice lacking Bmal1 (Arntl, Mop3), one of the central circadian clock genes. They found that both male and female Bmal1 knockout (KO) mice are infertile. Gross and microscopic inspection of the reproductive anatomy of both sexes suggested deficiencies in steroidogenesis. Male Bmal1 KO mice had low testosterone and high luteinizing hormone serum concentrations, suggesting a defect in testicular Leydig cells. Importantly, Leydig cells rhythmically express BMAL1 protein, suggesting peripheral control of testosterone production by this clock protein. Expression of steroidogenic genes was reduced in testes and other steroidogenic tissues of Bmal1 KO mice. In particular, expression of the steroidogenic acute regulatory protein (StAR) gene and protein, which regulates the rate-limiting step of steroidogenesis, was decreased in testes from Bmal1 KO mice. A direct effect of BMAL1 on StAR expression in Leydig cells was indicated by in vitro experiments showing enhancement of StAR transcription by BMAL1. Other hormonal defects in male Bmal1 KO mice suggest that BMAL1 also has functions in reproductive physiology outside of the testis. These results enhance understanding of how the circadian clock regulates reproduction.Keywords circadian rhythms; fertility; testosterone; testes; sperm; StAR; mice Disruption of circadian rhythms results in a variety of pathophysiologic states (Hastings et al., 2003). Reproductive physiology, in particular, is profoundly influenced by circadian rhythms (Boden and Kennaway, 2006). In various insect species, the circadian clock is necessary for proper ovulation, sperm production, and fertility (Giebultowicz et al., 1989;Beaver et al., 2002;Beaver et al., 2003;Beaver and Giebultowicz, 2004 (Lucas and Eleftheriou, 1980;Clair et al., 1985;Chappell et al., 2003;Miller et al., 2004). For example, the surge of luteinizing hormone (LH) necessary for ovulation in rodents, which occurs at the same time of day during each estrous cycle, requires a functional circadian clock (Barbacka-Surowiak et al., 2003). In addition, at the onset of puberty, a clear diurnal rhythm of gonadotropin serum levels is established in both mice and humans (Andrews and Ojeda, 1981;Jean-Faucher et al., 1986;Dunkel et al., 1992;Apter et al., 1993). It is unclear whether this diurnal rhythm continues into adulthood, but testosterone serum concentration shows daily oscillations in adult male mice and humans (Lucas and Eleftheriou, 1980;Clair et al., 1985). Although the association between circadian rhythms and testosterone is a long-established phenomenon, the molecular mechanisms by which the circadian clock regulates testosterone production are unknown.The circadian clock is based on a transcription translation feedback loop that results in the cyclic expression of genes and proteins over a 24-h p...

Assisted reproductive technologies (ART) are associated with several complications including low birth weight, abnormal placentation and increased risk for rare imprinting disorders. Indeed, experimental studies demonstrate ART procedures independent of existing infertility induce epigenetic perturbations in the embryo and extraembryonic tissues. To test the hypothesis that these epigenetic perturbations persist and result in adverse outcomes at term, we assessed placental morphology and methylation profiles in E18.5 mouse concepti generated by in vitro fertilization (IVF) in two different genetic backgrounds. We also examined embryo transfer (ET) and superovulation procedures to ascertain if they contribute to developmental and epigenetic effects. Increased placental weight and reduced fetal-to-placental weight ratio were observed in all ART groups when compared with naturally conceived controls, demonstrating that non-surgical embryo transfer alone can impact placental development. Furthermore, superovulation further induced overgrowth of the placental junctional zone. Embryo transfer and superovulation defects were limited to these morphological changes, as we did not observe any differences in epigenetic profiles. IVF placentae, however, displayed hypomethylation of imprinting control regions of select imprinted genes and a global reduction in DNA methylation levels. Although we did not detect significant differences in DNA methylation in fetal brain or liver samples, rare IVF concepti displayed very low methylation and abnormal gene expression from the normally repressed allele. Our findings suggest that individual ART procedures cumulatively increase placental morphological abnormalities and epigenetic perturbations, potentially causing adverse neonatal and long-term health outcomes in offspring.

Successful fertilization in humans, achieved when parental chromosomes intermix at first mitosis, requires centrosome restoration and microtubule-mediated motility. Imaging of inseminated human oocytes reveals that the sperm introduces the centrosome. The centrosome then nucleates the new microtubule assembly to form the sperm aster--a step essential for successful fertilization. Oocytes from some infertile patients failed to complete fertilization because of defects in uniting the sperm and egg nuclei, indicating that failure to properly effect the cytoplasmic motions uniting the nuclei results in human infertility. These discoveries have important implications for infertility diagnosis and managing reproduction.

Assisted reproductive technologies (ART) have enabled millions of couples with compromised fertility to conceive children. Nevertheless, there is a growing concern regarding the safety of these procedures due to an increased incidence of imprinting disorders, premature birth, and low birth weight in ART-conceived offspring. An integral aspect of ART is the oxygen concentration used during in vitro development of mammalian embryos, which is typically either atmospheric (~20%) or reduced (5%). Both oxygen tension levels have been widely used, but 5% oxygen improves preimplantation development in several mammalian species, including that of humans. To determine whether a high oxygen tension increases the frequency of epigenetic abnormalities in mouse embryos subjected to ART, we measured DNA methylation and expression of several imprinted genes in both embryonic and placental tissues from concepti generated by in vitro fertilization (IVF) and exposed to 5% or 20% oxygen during culture. We found that placentae from IVF embryos exhibit an increased frequency of abnormal methylation and expression profiles of several imprinted genes, compared to embryonic tissues. Moreover, IVF-derived placentae exhibit a variety of epigenetic profiles at the assayed imprinted genes, suggesting that these epigenetic defects arise by a stochastic process. Although culturing embryos in both of the oxygen concentrations resulted in a significant increase of epigenetic defects in placental tissues compared to naturally conceived controls, we did not detect significant differences between embryos cultured in 5% and those cultured in 20% oxygen. Thus, further optimization of ART should be considered to minimize the occurrence of epigenetic errors in the placenta.

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The establishment of pregnancy requires a successful molecular interaction between the trophectoderm cells of the blastocyst stage embryo and the endometrial cells of the uterus. These interactions are complex and require synchronous development and coordinated endocrine, paracrine, and autocrine communication. In this study, we demonstrate that the tetraspan protein epithelial membrane protein-2 (EMP2) is involved in these molecular interactions during implantation. EMP2, which is highly expressed in the uterus, translocates from an intracellular location to the apical surface of the endometrial epithelium during the window of implantation and is expressed in decidualized stromal cells. We developed plasmid constructs that utilized either ribozyme-mediated or short hairpin RNA-mediated mechanisms to target endometrial EMP2 mRNA for destruction. These constructs were transfected into the mouse uterus on day 1 of pregnancy using the technique of in vivo reproductive tract gene transfer. Reduction in EMP2 expression by either method resulted in a significant decrease in the number of implantation sites in the treated uterine horns as compared to control horns. These studies indicate a previously unknown function of tetraspan proteins in implantation and could provide a molecular framework for the development of therapeutic modalities for both contraception and fertility.