Cell-mediated immunity (CMI) is key to defense against intracellular pathogens such as Chlamydia trachomatis and viruses that infect the lower female genital tract, but little is known about CMI at this site. Recent studies indicate that there are immunological microenvironments within the female genital tract, and that immune functions are affected by hormones as well as infections and inflammatory processes. To determine the distribution of mediators of CMI within the lower female genital tract, we have enumerated and characterized T-lymphocyte subsets and natural killer and antigen presenting cells (APCs; macrophages and dendritic cells) in the introitus, vagina, ectocervix, endocervix and cervical transformation zone (TZ) from healthy women, and have examined the effects of the menstrual cycle, menopause and inflammation on these parameters. In women without inflammation, T cells and APCs were most prevalent in the cervical TZ and surrounding tissue. Intraepithelial lymphocytes were predominantly CD8+ T cell+; most CD8+ cells in the TZ and endocervix, and a proportion of cells in the ectocervix, expressed T-cell internal antigen-1, a marker of cytotoxic potential. In contrast, the normal vaginal mucosa contained few T cells and APCs. Cervicitis and vaginitis cases had increased numbers of intraepithelial CD8+ and CD4+ lymphocytes and APCs. The menstrual cycle and menopause had no apparent effect on cellular localization or abundance in any of the lower genital tract tissues. These data indicate that the cervix, especially the TZ, is the major inductive and effector site for CMI in the lower female genital tract. Because CD4+ T cells and APCs are primary host cells for human immunodeficiency virus type 1 (HIV-1), these data also provide further evidence that the cervix is a primary infection site of HIV-1, and that inflammation increases the risk of HIV transmission. Abstract Regional variations in location and abundance of subpopulations of T-lymphocytes and antigen-presenting cells occur in the lower female genital tract and are unaffected by the menstrual cycle but are dramatically affected by inflammation
Spermatogonial stem cells are the only stem cells in the body that transmit genetic information to the next generation. These cells can be cultured for extended periods in the presence of serum and feeder cells. However, little is known about factors that regulate self-renewal division of spermatogonial stem cells. In this investigation we examined the possibility of establishing culture systems for spermatogonial stem cells that lack serum or a feeder cell layer. Spermatogonial stem cells could expand in serum-free conditions on mouse embryonic fibroblasts (MEFs), or were successfully cultivated without feeder cells on a laminin-coated plate. However, they could not expand when both serum and feeder cells were absent. Although the cells cultured on laminin differed phenotypically from those on feeder cells, they grew exponentially for at least 6 mo, and produced normal, fertile progeny following transplantation into infertile mouse testis. This culture system will provide a new opportunity for understanding the regulatory mechanism that governs spermatogonial stem cells. Abstract Mouse male germline stem cells can be cultured without serum or feeder cells
MicroRNAs play important roles in regulating development at both transcriptional and posttranscriptional levels. Here, we report 29 microRNAs from mouse testis that are differentially expressed as the prepubertal testis differentiates to the adult testis. Using computational analyses to identify potential microRNA target mRNAs, we identify several possible male germ cell target mRNAs. One highly conserved sequence in the 3â²-untranslated region (UTR) of transition protein 2 ( Tnp2 ) mRNA, a testis-specific and posttranscriptionally regulated mRNA in postmeiotic germ cells, is complementary to Mirn122a. Mirn122a is enriched in late-stage male germ cells and is predominantly on polysomes. Mirn122a, but not another noncomplementary microRNA, inhibits the activity of a luciferase reporter construct containing the 3â²-UTR of Tnp2. Site-directed mutations of Mirn122a indicate that base pairing of the 5â²-region of Mirn122a to its complementary site in the 3â²-UTR of Tnp2 mRNA is essential for the downregulation of luciferase activity. Real-time reverse transcription-polymerase chain reaction and ribonuclease protection assays reveal that the Mirn122a -directed decrease of the Tnp2 reporter gene activity results from mRNA cleavage. We propose that specific microRNAs, such as Mirn122a, could be involved in the posttranscriptional regulation of mRNAs such as Tnp2 in the mammalian testis. Abstract Mirn122a, a microRNA enriched in late-stage male germ cells, can inhibit Tnp2 expression by directing the cleavage of its mRNA
There are two estrogen receptor (ER) subtypes in fish, Esr1 and Esr2 (formerly ERÎ± and ERÎ²), and in some species the Esr2 subtype has two forms, Esr2b (formerly ERÎ²1) and Esr2a (formerly ERÎ²2 or ERÎ³). There is little information, however, on the different characteristics and functional significance of the two receptor subtypes in fish, and this is especially relevant for understanding the disruption of ER signaling by chemicals with estrogenic activity. In this study, the full-length cDNAs for esr1 (3167 base pairs [bp]) and esr2b (2318 bp), and a partial-length (267 bp) cDNA for esr2a, were cloned and characterized in fathead minnow (fhm; Pimephales promelas ), and their patterns of expression established during development and in adults. Real-time polymerase chain reaction revealed some clear distinctions in the ontogenic and tissue expression of fhm esr1, esr2b, and esr2a, suggesting different functions for each ER subtype. Fhm ERs were expressed in brain, pituitary, liver, gonad, intestine, and gill of male and female fish, esr2b and esr2a were also expressed in muscle. Fhm esr1 and esr2b were expressed predominantly in the liver, whereas fhm esr2a was expressed predominantly in intestine and was lowest expressed in liver. Responses of the different hepatic ERs in male fathead minnow exposed to 100 ng estradiol/L differed, with a significant induction (5-fold) of fhm esr1 but no effect on esr2b or esr2a expression, suggesting different mechanisms of regulation for the different ERs. The detailed characterization of ERs in fathead minnow provides the foundation for understanding the molecular basis of estrogenic disruption in fish. Abstract The cloning and distinct expression profiles of estrogen receptors esr1, esr2a and esr2b in a teleost species provide clues into the roles of the different Esr's in reproduction in fish
Developmental exposure to estrogenic chemicals induces morphological, functional, and behavioral anomalies associated with reproduction. Humans are routinely exposed to bisphenol-A (BPA), an estrogenic compound that leaches from dental materials and plastic food and beverage containers. The aim of the present study was to determine the effects of in utero exposure to low, environmentally relevant doses of BPA on the development of female reproductive tissues in CD-1 mice. In previous publications, we have shown that this treatment alters the morphology of the mammary gland and affects estrous cyclicity. Here we report that in utero exposure to 25 and 250 ng BPA/ kg of body weight per day via osmotic pumps implanted into pregnant dams at Gestational Day 9 induces alterations in the genital tract of female offspring that are revealed during adulthood. They include decreased wet weight of the vagina, decreased volume of the endometrial lamina propria, increased incorporation of bromodeoxyuridine into the DNA of endometrial gland epithelial cells, and increased expression of estrogen receptor-Î± (ERÎ±) and progesterone receptor in the luminal epithelium of the endometrium and subepithelial stroma. Because ERÎ± is known to be expressed in these estrogen-target organs at the time of BPA exposure, it is plausible that BPA may directly affect the expression of ER-controlled genes involved in the morphogenesis of these organs. In addition, BPA-induced alterations that specifically affect hypothalamic-pituitary-gonadal axis function may further contribute to the anomalies observed at 3 mo of age, long after the cessation of BPA exposure. Abstract Prenatal exposure to BPA induces alterations in the female genital tract revealed during adulthood
Male reproductive success is determined by the ability of males to gain sexual access to females and by their ability to fertilize ova. Among polygynous mammals, males differ markedly in their reproductive success, and a great deal of effort has been made to understand how selective forces have shaped traits that enhance male competitiveness both before and after copulation (i.e., sperm competition). However, the possibility that males also may differ in their fertility has been ignored under the assumption that male infertility is rare in natural populations because selection against it is likely to be strong. In the present study, we examined which semen traits correlate with male fertility in natural populations of Iberian red deer ( Cervus elaphus hispanicus ). We found no trade-offs between semen traits. Our analyses revealed strong associations between sperm production and sperm swimming velocity, sperm motility and proportion of morphologically normal spermatozoa, and sperm viability and acrosome integrity. These last two variables had the lowest coefficients of variation, suggesting that these traits have stabilized at high values and are unlikely to be related to fitness. In a fertility trial, our results show a large degree of variation in male fertility, and differences in fertility were determined mainly by sperm swimming velocity and by the proportion of morphologically normal sperm. We conclude that male fertility varies substantially in natural populations of Iberian red deer and that, when sperm numbers are equal, it is determined mainly by sperm swimming velocity and sperm morphology. Abstract Male fertility in natural populations of red deer is determined by sperm velocity and the proportion of normal spermatozoa
Maturation of spermatozoa, including the acquisition of motility and the ability to undergo capacitation, occurs during transit through the dynamic environment of the epididymis. The microenvironments created along the length of the epididymal tubule are essential to the molecular modifications of spermatozoa that result in fertile gametes. The secretory and resorptive processes of the epithelial cells that line this tubule generate these microenvironments. In the current study, 10 morphologically distinct segments of the mouse epididymis were identified by microdissection. We hypothesized that the changing environments of the epididymal lumen are established by differential gene expression among these segments. RNA isolated from each of the 10 segments was analyzed by microarray analysis. More than 17â000 genes are expressed in the mouse epididymis, compared with about 12â000 genes identified from whole epididymal samples. Screening a panel of normal mouse tissues identified both epididymal-selective and epididymal-specific transcripts. In addition, this study identified 2168 genes that are up-regulated or down-regulated by greater than 4-fold between at least two different segments. The expression patterns of these genes identify distinct patterns of segmental regulation. Using principal component analysis, we determined that the 10 segments form 6 different transcriptional units. These analyses elucidate the changes in gene expression along the length of the epididymis for 17â000 expressed transcripts and provide a powerful resource for the research community in future studies of the biological factors that mediate epididymal sperm maturation. Abstract Proteomic analyses elucidate changes in gene expression along the length of the epididymis for 17000 expressed transcripts and provide a powerful resource for the research community in future studies of the biological factors that mediate epididymal sperm maturation
In the present study, we cloned and characterized zebrafish FSH receptor (Fshr) and LH receptor (Lhr). Both fshr and lhr were abundantly expressed in the zebrafish gonads; however, they could also be detected in the kidney and liver, respectively. When overexpressed in mammalian cell lines together with a cAMP-responsive reporter gene, zebrafish Fshr responded to goldfish pituitary extract but not hCG, whereas Lhr could be activated by both. It was further demonstrated that Fshr was specific to bFSH, while Lhr could be stimulated by both bovine FSH and LH. Low level of fshr expression could be detected in the immature ovary, but the level steadily increased during vitellogenesis of the first cohort of developing follicles. In contrast, the expression of lhr could barely be detected in the immature ovary, but it became detectable at the beginning of vitellogenesis and steadily increased afterward with the peak level reached at the full-grown stage. At the follicle level, the expression of fshr was very weak in the follicles of primary growth stage but significantly increased with the follicles entering vitellogenesis. However, after reaching the maximal level in the midvitellogenic follicles, the level of fshr expression dropped slightly but significantly at the full-grown stage. in comparison, the expression of lhr obviously lagged behind that of fshr. Its expression became detectable only when the follicles started to accumulate yolk granules, but the level rose steadily afterward and reached the peak at the full-grown stage before oocyte maturation. These results suggest differential roles for Fshr and Lhr in zebrafish ovarian follicle development.
The application of microarray technology to the study of mammalian organogenesis can provide greater insights into the steps necessary to elicit a functionally competent tissue. To this end, a temporal profile of gene expression was generated with the purpose of identifying changes in gene expression occurring within the developing male and female embryonic gonad. Gonad tissue was collected from mouse embryos at 11.5, 12.5, 14.5, 16.5, and 18.5 days postcoitum (dpc) and relative steady-state levels of mRNA were determined using the Affymetrix MGU74v2 microarray platform. Statistical analysis produced 3693 transcripts exhibiting differential expression during male and/or female gonad development. At 11.5 dpc, the gonad is morphologically indifferent, but at 12.5 dpc, transitions to a male or female phenotype are discernible by the appearance of testicular cords. A number of genes are expressed during this period and many share similar expression profiles in both sexes. As expected, the expression of two well-known sex determination genes, specifically Sry and Sox9, is unique to the testis. Beyond 12.5 dpc, differential gene expression becomes increasingly evident as the male and female tissue morphologically and physiologically diverges. This is evident by two unique waves of transcriptional activity occurring after 14.5 dpc in the male and female. With this study, a large number of transcripts comprising the murine transcriptome can be examined throughout male and female embryonic gonad development and allow for a more complete description of gonad differentiation and development. Abstract The differences in gene expression during the development of the gonad and between male and female gonads from 11.5 dpc to birth are revealed
In the present study, we cloned and characterized zebrafish FSH receptor (Fshr) and LH receptor (Lhr). Both fshr and lhr were abundantly expressed in the zebrafish gonads; however, they could also be detected in the kidney and liver, respectively. When overexpressed in mammalian cell lines together with a cAMP-responsive reporter gene, zebrafish Fshr responded to goldfish pituitary extract but not hCG, whereas Lhr could be activated by both. It was further demonstrated that Fshr was specific to bFSH, while Lhr could be stimulated by both bovine FSH and LH. Low level of fshr expression could be detected in the immature ovary, but the level steadily increased during vitellogenesis of the first cohort of developing follicles. In contrast, the expression of lhr could barely be detected in the immature ovary, but it became detectable at the beginning of vitellogenesis and steadily increased afterward with the peak level reached at the full-grown stage. At the follicle level, the expression of fshr was very weak in the follicles of primary growth stage but significantly increased with the follicles entering vitellogenesis. However, after reaching the maximal level in the midvitellogenic follicles, the level of fshr expression dropped slightly but significantly at the full-grown stage. In comparison, the expression of lhr obviously lagged behind that of fshr. Its expression became detectable only when the follicles started to accumulate yolk granules, but the level rose steadily afterward and reached the peak at the full-grown stage before oocyte maturation. These results suggest differential roles for Fshr and Lhr in zebrafish ovarian follicle development.
Male reproductive tract abnormalities associated with testicular dysgenesis in humans also occur in male rats exposed gestationally to some phthalate esters. We examined global gene expression in the fetal testis of the rat following in utero exposure to a panel of phthalate esters. Pregnant Sprague-Dawley rats were treated by gavage daily from Gestational Days 12 through 19 with corn oil vehicle (1 ml/kg) or diethyl phthalate (DEP), dimethyl phthalate (DMP), dioctyl tere-phthalate (DOTP), dibutyl phthalate (DBP), diethylhexyl phthalate (DEHP), dipentyl phthalate (DPP), or benzyl butyl phthalate (BBP) at 500 mg/kg per day. Testes were isolated on Gestational Day 19, and global changes in gene expression were determined. Of the approximately 30â000 genes queried, expression of 391 genes was significantly altered following exposure to the developmentally toxic phthalates (DBP, BBP, DPP, and DEHP) relative to the control. The developmentally toxic phthalates were indistinguishable in their effects on global gene expression. No significant changes in gene expression were detected in the nondevelopmentally toxic phthalate group (DMP, DEP, and DOTP). Gene pathways disrupted include those previously identified as targets for DBP, including cholesterol transport and steroidogenesis, as well as newly identified pathways involved in intracellular lipid and cholesterol homeostasis, insulin signaling, transcriptional regulation, and oxidative stress. Additional gene targets include alpha inhibin, which is essential for normal Sertoli cell development, and genes involved with communication between Sertoli cells and gonocytes. The common targeting of these genes by a select group of phthalates indicates a role for their associated molecular pathways in testicular development and offers new insight into the molecular mechanisms of testicular dysgenesis. Abstract The common targeting by reproductively toxic phthalates of molecular networks associated with testosterone production and interaction between Sertoli cells and gonocytes in the developing fetal testis implicates these gene pathways in the etiology of testicular dysgenesis
The overall understanding of the sex differentiation cascade in vertebrates is still growing slowly, probably because of the variety of vertebrate models used and the number of molecular players yet to be discovered. Finding conserved mechanisms among vertebrates should provide a better view of the key factors involved in this process. To this end, we used real-time reverse transcription-polymerase chain reaction to produce a temporal map of fluctuations in mRNA expression of 102 genes during sex differentiation and early gametogenesis in the rainbow trout ( Oncorhynchus mykiss ). We used these 102 temporal gene expression patterns as a basis for a hierarchical clustering analysis to find characteristic clusters of coexpressed genes. Analysis of some of these gene clusters suggested a conserved overall expression profile between the sex differentiation cascade in fish and mammals. Among these conserved molecular mechanisms, sox9, dmrt1, amh, nr5a1, nr0b1, igf1, and igf1ra are, for instance, characterized as early expressed genes involved in trout testicular differentiation as it is known or suggested in mammals. On the contrary, foxl2, fst, and lhr are characterized as early expressed genes during trout ovarian differentiation, as also found in mammals. Apart from this high conservation, our analysis suggests some potential new players, such as the fshb subunit gene, which is detected here for the first time, to our knowledge, in the female differentiating gonad of a vertebrate species and displays a specific overexpression that coincides in timing with the occurrence of first oocyte meioses, or the pax2 gene, which displays an early and testis-specific expression profile. Abstract Gene profiles during gonadal sex differentiation and early gametogenesis in trout indicate conservation of the sex differentiation cascade in vertebrates and reveal potential new molecular players in sex differentiation
Gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) play critical roles in vertebrate reproduction. In the present study, we cloned and characterized zebrafish FSH beta (fshb), LH beta (lhb), and GTH alpha (cga) subunits. Compared with the molecules of other teleosts, the cysteine residues and potential glycosylation sites are fully conserved in zebrafish Lhb and Cga but not in Fshb, whose cysteines exhibit unique distribution. Interestingly, in addition to the pituitary, fsh beta, Ih beta, and cga were also expressed in some extrapituitary tissues, particularly the gonads and brain. In situ hybridization showed that zebrafish fsh beta and lh beta were expressed in two distinct populations of gonadotrophs in the pituitary. Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that all the three subunits increased expression before ovulation (0100-0400) when the germinal vesicles in the full-grown follicles were migrating toward the periphery, but the levels dropped at 0700, when ovulation occurred. Recombinant zebrafish FSH (zfFSH) and LH (zfLH) were produced in the Chinese hamster ovary (CHO) cells and their effects on the cognate receptors (zebrafish Fshr and Lhr) tested. Interestingly, zfFSH specifically activated zebrafish Fshr expressed together with a cAMP-responsive reporter gene in the CHO cells, whereas zfLH could stimulate both Fshr and Lhr. In conclusion, the present study systematically investigated gonadotropins in the zebrafish in terms of their structure, spatial-temporal expression patterns, and receptor specificity. These results, together with the availability of recombinant zfFSH and zfLH, provide a solid foundation for further studies on the physiological relevance of FSH and LH in the zebrafish, one of the top biological models in vertebrates.
The extent, causes, and physiological significance of the variation in number of follicles growing during ovarian follicular waves in human beings and cattle are unknown. Therefore, the present study examined the variability and repeatability in numbers of follicles 3 mm or greater in diameter during the follicular waves in bovine estrous cycles, and we determined if the variation in number of follicles during waves was associated with alterations in secretion of FSH, estradiol, inhibin, and insulin-like growth factor I (IGF-I). Dairy cattle were subjected to twice-daily ultrasound analysis to count total number of antral follicles 3 mm or greater in diameter throughout 138 different follicular waves. In another study, blood samples were taken at frequent intervals from cows that consistently had low or very high numbers of follicles during waves and were subjected to immunoassays. Results indicate the following: First, despite an approximately sevenfold variation in number of follicles during waves among animals and marked differences in age, stage of lactation, and season of the year, a very highly repeatable (0.95) number of follicles 3 mm or greater in diameter is maintained during the ovulatory and nonovulatory follicular waves of individuals. Second, variation in number of follicles 3 mm or greater in diameter during waves and the inverse association of number of follicles during waves with FSH are not directly explained by alterations in the patterns of secretion of estradiol, inhibin, or IGF-I. Third, ovarian ultrasound analysis can be used reliably by investigators to identify cattle that consistently have low or high numbers of follicles during waves, thus providing a novel experimental model to determine the causes and physiological significance of the high variation in antral follicle number during follicular waves among single-ovulating species, such as cattle or humans. Abstract Number of antral follicles during follicular waves is highly repeatable in individual cattle
Spermatogonial stem cells (SSCs) are essential for spermatogenesis, and these adult tissue stem cells balance self-renewal and differentiation to meet the biological demand of the testis. The developmental dynamics of SSCs are controlled, in part, by factors in the stem cell niche, which is located on the basement membrane of seminiferous tubules situated among Sertoli cells. Sertoli cells produce glial cell line-derived neurotrophic factor (GDNF), and disruption of GDNF expression results in spermatogenic defects and infertility. The GDNF signals through a receptor complex that includes GDNF family receptor Î±1 (GFRA1), which is thought to be expressed by SSCs. However, expression of GFRA1 on SSCs has not been confirmed by in vivo functional assay, which is the only method that allows definitive identification of SSCs. Therefore, we fractionated mouse pup testis cells based on GFRA1 expression using magnetic activated cell sorting. The sorted and depleted fractions of GFRA1 were characterized for germ cell markers by immunocytochemistry and for stem cell activity by germ cell transplantation. The GFRA1-positive cell fraction coeluted with other markers of SSCs, including ITGA6 and CD9, and was significantly depleted of KIT-positive cells. The transplantation results confirmed that a subpopulation of SSCs expresses GFRA1, but also that the stem cell pool is heterogeneous with respect to the level of GFRA1 expression. Interestingly, POU5F1-positive cells were enriched nearly 15-fold in the GFRA1-selected fraction, possibly suggesting heterogeneity of developmental potential within the stem cell pool. Abstract Spermatogonial stem cells can be enriched using magnetic activated cell sorting with GFRA1 antibodies, revealing a distinct GFRA1-positive subpopulation in the mouse pup testis that coexpresses the pluripotency marker POU5F1
A search for genes expressed more highly in mouse cumulus cells than mural granulosa cells by subtraction hybridization yielded Slc38a3. SLC38A3 is a sodium-coupled neutral amino acid transporter having substrate preference for l -glutamate, l -histidine, and l -alanine. Detectable levels of Slc38a3 mRNA were found by in situ hybridization in granulosa cells of large preantral follicles, but levels were higher in all granulosa cells of small antral follicles; expression became limited to cumulus cells of large antral follicles. Expression of Slc38a3 mRNA in granulosa cells was promoted by fully grown oocytes from antral follicles but not by growing oocytes from preantral follicles. Fully grown oocytes were dependent on cumulus cells for uptake of l -alanine and l -histidine but not l -leucine. Fully grown but not growing oocytes secreted one or more paracrine factors that promoted cumulus cell uptake of all three amino acids but of l -alanine and l -histidine to a much greater extent than l -leucine. Uptake of l -leucine appeared dependent primarily on contact-mediated signals from fully grown oocytes. Fully grown oocytes also promoted elevated levels of Slc38a3 mRNA and l -alanine transport by preantral granulosa cells, but growing oocytes did not. Therefore, fully grown oocytes secrete one or more paracrine factors that promote cumulus cell uptake of amino acids that oocytes themselves transport poorly. These amino acids are likely transferred to oocytes via gap junctions. Thus, oocytes use paracrine signals to promote their own development via metabolic cooperativity with cumulus cells. The ability of oocytes to mediate this cooperativity is developmentally regulated and acquired only in later stages of oocyte development. Abstract Mouse oocytes regulate the uptake of amino acids by cumulus cells, and oocyte competence to carry out this function is developmentally regulated.
The present study sought to assess the combined effects of body composition and diet (level of feeding) on the postfertilization developmental potential of oocytes recovered from heifers using ultrasound-guided transvaginal follicular aspiration and to relate oocyte quality to the metabolic status of these animals. By collecting oocytes on repeated occasions spanning several weeks, it was possible to assess the cumulative effects of changes in nutritional status on oocyte quality over this period. Twenty-four heifers of low and moderate body condition were placed on one of two levels of feeding (equivalent to once or twice the maintenance requirements of these animals). Oocytes were recovered at two defined time points within each of three successive estrous cycles and were matured, fertilized, and cultured to the blastocyst stage in vitro. The results show that the effect of feeding level on oocyte quality is dependent on the body condition of the animal, with the high level of feeding being beneficial to oocytes from animals of low body condition but detrimental to oocytes from animals of moderately high body condition. Furthermore, the effects of high levels of feeding on oocyte quality were cumulative, with blastocyst yields for relatively fat heifers on twice the maintenance requirement deteriorating with time relative to yields for relatively thin heifers on the same level of feeding. Finally, a significant proportion of the moderately fat animals on the high level of feeding were hyperinsulinemic, and we show, to our knowledge for the first time in ruminants, that this condition is associated with impaired oocyte quality. Abstract The cumulative effects of diet on oocyte quality in cattle are dependant on body composition
Human embryonic stem (hES) cells are usually established and maintained on mouse embryonic fibroblast (MEFs) feeder layers. However, it is desirable to develop human feeder cells because animal feeder cells are associated with risks such as viral infection and/or pathogen transmission. In this study, we attempted to establish new hES cell lines using human uterine endometrial cells (hUECs) to prevent the risks associated with animal feeder cells and for their eventual application in cell-replacement therapy. Inner cell masses (ICMs) of cultured blastocysts were isolated by immunosurgery and then cultured on mitotically inactivated hUEC feeder layers. Cultured ICMs formed colonies by continuous proliferation and were allowed to proliferate continuously for 40, 50, and 55 passages. The established hES cell lines (Miz-hES-14, -15, and -9, respectively) exhibited typical hES cells characteristics, including continuous growth, expression of specific markers, normal karyotypes, and differentiation capacity. The hUEC feeders have the advantage that they can be used for many passages, whereas MEF feeder cells can only be used as feeder cells for a limited number of passages. The hUECs are available to establish and maintain hES cells, and the high expression of embryotrophic factors and extracellular matrices by hUECs may be important to the efficient growth of hES cells. Clinical applications require the establishment and expansion of hES cells under stable xeno-free culture systems. Abstract Endometrial cells can substitute for fibroblasts as feeder cells for human ES cells
Mammalian sperm acquire fertilization capacity after residing in the female tract during a process known as capacitation. The present study examined whether cholesterol efflux during capacitation alters the biophysical properties of the sperm plasma membrane by potentially reducing the extent of lipid raft domains as analyzed by the isolation of detergent-resistant membrane fractions using sucrose gradients. In addition, this work investigated whether dissociation of the detergent-resistant membrane fraction during capacitation alters resident sperm raft proteins. Mouse sperm proteins associated with such fractions were studied by silver staining, tandem mass spectrometry, and Western blot analysis. Caveolin 1 was identified in sperm lipid rafts in multimeric states, including a high-molecular-weight oligomer that is sensitive to degradation under reducing conditions at high pH. Capacitation resulted in reduction of the light buoyant-density, detergent-resistant membrane fraction and decreased the array of proteins isolated within this fraction, including loss of the high-molecular-weight caveolin 1 oligomers. Proteomic analysis of sperm proteins isolated in the light buoyant-density fraction identified several proteins, including hexokinase 1, testis serine proteases 1 and 2, TEX101, hyaluronidase (PH20, SPAM1), facilitated glucose transporter 3, lactate dehydrogenase A, carbonic anhydrase IV, IZUMO, pantophysin, basigin, and cysteine-rich inhibitory secretory protein 1. Capacitation also resulted in a significant reduction of sperm labeling by the fluorescent lipid-analog DiIC16, indicating that capacitation alters the liquid-ordered domains in the sperm plasma membrane. The observations that capacitation alters the protein composition of the detergent-resistant membrane fractions is consistent with the hypothesis that cholesterol efflux during capacitation dissociates lipid raft constituents, initiating signaling events that lead to sperm capacitation. Abstract More than 25 proteins were identified in sperm lipid raft domains by tandem mass spectrometry analysis, and sperm capacitation was correlated with disruption of these domains
Abstract Outbred female CD-1 mice were treated with genistein (Gen), the primary phytoestrogen in soy, by s.c. injections on Neonatal Days 1–5 at doses of 0.5, 5, or 50 mg/kg per day (Gen-0.5, Gen-5, and Gen-50). The day of vaginal opening was observed in mice treated with Gen and compared with controls, and although there were some differences, they were not statistically significant. Gen-treated mice had prolonged estrous cycles with a dose- and age-related increase in severity of abnormal cycles. Females treated with Gen-0.5 or Gen-5 bred to control males at 2, 4, and 6 mo showed statistically significant decreases in the number of live pups over time with increasing dose; at 6 mo, 60% of the females in the Gen-0.5 group and 40% in the Gen-5 group delivered live pups compared with 100% of controls. Mice treated with Gen-50 did not deliver live pups. At 2 mo, >60% of the mice treated with Gen-50 were fertile as determined by uterine implantation sites, but pregnancy was not maintained; pregnancy loss wa...