Reproductive Tract Tissues Research Articles

411. Macrophage abundance and phenotype in the mammary gland fluctuates throughout the estrous cycle

Macrophages are versatile cells with roles in the generation and execution of immune responses, tissue breakdown and remodelling and production of regulatory cytokines. These cells are implicated in development of reproductive tract tissues that mature after birth. Descriptive and knockout studies indicates that macrophages play multiple roles in epithelial cell proliferation, phagocytosis and tissue remodelling during pubertal mammary gland development, pregnancy, lactation, and involution. However, the role of macrophages in remodelling over the course of the ovarian cycle has not been previously investigated. Oestrus cycles were monitored in C57/Bl6 mice by daily vaginal smears, and mammary glands were analysed for the abundance and location of macrophages in all four stages of the cycle (n = 6–8 per group). Absolute number of macrophages was quantified by FACs analysis of digested mammary gland tissue stained with macrophage-specific antibody F4/80. The location of macrophages was investigated in fixed, paraffin embedded tissue by immunohistochemistry using F4/80 antibody. H&E staining revealed that the most differentiated epithelial ducts and alveoli structures appeared in diestrus and proestrus. Whilst there was no difference in the absolute number of macrophages at any stage, the location of these macrophages varied. Macrophages accumulated around the more highly differentiated epithelial ducts, leading to significantly more macrophages around the ducts during proestrus compared with metestrus (P < 0.05). These macrophages were in close contact with the epithelium, and displayed long projections and widespread cytoplasm. In contrast, macrophages on the edge of the gland within the collagen-rich connective tissue were characteristically round in shape. These results indicate that macrophages are present in the adult virgin mammary gland, and fluctuate in location and phenotype over the course of the oestrous cycle. Further analysis of the phenotypes of macrophages present in these different locations is required for a more comprehensive understanding of the role of macrophages in mammary tissue homeostasis.

Female reproductive tract fluids: composition, mechanism of formation and potential role in the developmental origins of health and disease

The oviduct and uterus provide the environments for the earliest stages of mammalian embryo development. However, little is known about the mechanisms that underlie the formation of oviduct and uterine fluids, or the extent to which the supply of nutrients via these reproductive tract tissues matches the nutrient requirements of early embryos. After reviewing our limited knowledge of these phenomena, a new experimental paradigm is proposed in which the epithelia lining the endosalpinx and endometrium are seen as the final components in a supply line that links maternal diet at one end and embryo uptake of nutrients at the other. When considered in this way, the oviduct and uterine epithelia become, for a few days, potentially the most critical maternal tissues in the establishment of a healthy pregnancy. In fulfilling this 'gatekeeper' role, female reproductive tract fluids have a key role in the 'developmental origins of health and disease' concept.

Susceptibility of Human Female Primary Genital Epithelial Cells to Herpes Simplex Virus, Type-2 and the Effect of TLR3 Ligand and Sex Hormones on Infection1

Genital epithelial cells (ECs) are the first line of defense that sexually transmitted viruses encounter. The mechanism of viral pathogenesis in these cells is not well understood. Here, we show that a primary cell culture model from human reproductive tract tissues can be used as a novel ex vivo model in examining the interaction of herpes simplex virus, type 2 (HSV-2), with female genital mucosa. Confluent, polarized primary cultures of human endometrial and cervical ECs were established and shown to be free from any significant contamination of any other cell type. Both endometrial and cervical ECs were found to be highly susceptible to HSV-2 infection. The kinetic of infection was similar to in vivo infection, with the earliest viral shedding seen at 18 h postinfection. Primary EC monolayers could be infected both apically and basolaterally, but preferential viral shedding was seen on the apical side of cells. Following treatment of the monolayers with poly (I:C), an innate immune activator that acts via TLR3, viral shedding was reduced significantly, comparable to levels seen when an antiviral formulation, acyclovir, was used. Treatment of epithelial and stromal co-cultures with estradiol increased HSV-2 infection in endometrial ECs, but viral shedding decreased following treatment with progesterone. To the best of our knowledge, this is the first study that examines the interaction of primary human female genital ECs with HSV-2, using an ex vivo culture model. The results provide valuable information regarding the susceptibility of women's genital ECs to HSV-2 and the ability of innate immunity and hormones to modify this susceptibility.

Long-term adverse effects of neonatal exposure to bisphenol A on the murine female reproductive tract

The developing fetus is uniquely sensitive to perturbation by chemicals with hormone-like activity. The adverse effects of prenatal diethylstilbestrol (DES) exposure are a classic example. Since concern has been mounting regarding the human health and environmental effects of bisphenol A (BPA), a high-production-volume chemical with estrogenic activity used in the synthesis of plastics, we investigated its long-term effects in an experimental animal model that was previously shown useful in studying the adverse effects of developmental exposure to DES. Outbred female CD-1 mice were treated on days 1–5 with subcutaneous injections of BPA (10, 100 or 1000 μg/kg/day) dissolved in corn oil or corn oil alone (Control). At 18 months, ovaries and reproductive tract tissues were examined. There was a statistically significant increase in cystic ovaries and cystic endometrial hyperplasia (CEH) in the BPA-100 group as compared to Controls. Progressive proliferative lesion (PPL) of the oviduct and cystic mesonephric (Wolffian) duct remnants were also seen in all of the BPA groups. More severe pathologies of the uterus following neonatal BPA treatment included adenomyosis, leiomyomas, atypical hyperplasia, and stromal polyps. These data suggest that BPA causes long-term adverse effects if exposure occurs during critical periods of differentiation.

The role of the endocannabinoid system in gametogenesis, implantation and early pregnancy

Maternal use of marijuana, in which the exocannabinoid Delta(9)-tetrahydrocannabinol is the most active psychoactive ingredient, is known to have adverse effects on various aspects of reproduction including ovulation, spermatogenesis, implantation and pregnancy duration. Endogenous cannabinoids of which Anandamide is the prototype are widely distributed in the body especially in the reproductive tract and pregnancy tissues and act through the same receptors as the receptor as Delta(9)-tetrahydrocannabinol. Anandamide, has been reported to have pleiotropic effects on human reproduction and in experimental animal models. It appears to be the important neuro-cytokine mediator synchronizing the embryo-endometrial development for timed implantation, the development of the embryo into the blastocyst and transport of the embryo across the fallopian tubes. The mechanisms by which it exerts these effects are unclear but could be via direct actions on the various sites within the reproductive system or its differential actions on vascular tone dependent. In this review article we bring together the current knowledge on the role of endoccanabinoids in reproduction and postulate on the potential mechanisms on how these affect reproduction. In addition, we examine its role on the endothelium and vascular smooth muscle as a potential mechanism for adverse pregnancy outcome.

GM-CSF regulation of embryo development and pregnancy

The reproductive tissues undergo profound structural changes and major immune adaptation to accommodate pregnancy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of an array of cytokines with pivotal roles in embryo implantation and subsequent development. Several cell lineages in the reproductive tract and gestational tissues synthesise GM-CSF under direction by ovarian steroid hormones and signalling agents originating in male seminal fluid and the conceptus. The pre-implantation embryo, invading placental trophoblast cells and the abundant populations of leukocytes controlling maternal immune tolerance are all subject to GM-CSF regulation. GM-CSF deficiency in pregnancy adversely impacts fetal and placental development, as well as progeny viability and growth after birth, highlighting this cytokine as a central maternal determinant of pregnancy outcome with clinical relevance in human fertility.

Identification of a Sensitive Period for Developmental Programming That Increases Risk for Uterine Leiomyoma in Eker Rats

Epidemiological and experimental animal studies have shown that exposure to xenoestrogens during reproductive tract development reprograms target tissues, leading to increased disease risk later in adult life. To understand what defines the critical risk period for this effect, termed developmental programming, the authors assess the sensitivity of the female reproductive tract to developmental programming during various stages of neonatal development. Eker rats, which are predisposed to develop uterine leiomyoma because of a germ-line defect in the tuberous sclerosis complex 2 (Tsc-2) tumor suppressor gene, were exposed to the xenoestrogen diethylstilbestrol (DES) on either postnatal days 3 to 5, 10 to 12, or 17 to 19, 3 important periods of reproductive tract development and differentiation. Developmental programming was observed in both carrier (Tsc-2(Ek/+)) and wild-type (Tsc-2(+/+)) rats exposed to DES at days 3 to 5 and days 10 to 12 but not in rats exposed at days 17 to 19. Developmental programming resulted in increased tumor suppressor gene penetrance in Tsc-2(Ek/+) females relative to vehicle controls. In contrast, DES exposure at days 17 to 19 did not significantly increase the incidence of uterine leiomyoma in carrier females, indicating that the window of susceptibility had closed by this time. Gene expression analysis to determine what defined the susceptible (days 3-5 and days 10-12) versus resistant (days 17-19) periods revealed that in adult myometrium, expression of the estrogen-responsive genes calbindin D(9)K and progesterone receptor had been reprogrammed in females exposed to DES at days 3 to 5 and days 10 to 12 but not in those exposed at days 17 to 19. Reprogramming in response to DES exposure resulted in a hyperresponsiveness to ovarian hormones and could be prevented by ovariectomy prior to sexual maturity. Furthermore, in the neonatal uterus, DES was equally effective at inducing transcription of estrogen-responsive genes during both sensitive and resistant periods, indicating that resistance to developmental programming was not due to an inability of the estrogen receptor to transactivate gene expression. Interestingly, the resistant period coincided with the time at which reproductive tract tissues are exposed to endogenous estrogen, suggesting that target tissues are most vulnerable to developmental programming during the period in which they would normally be maintained in an estrogen-naïve state.

Identification, cloning and functional characterization of novel sperm associated antigen 11 (SPAG11) isoforms in the rat

BackgroundSperm binding proteins and their C-terminal peptides of the Sperm Associated Antigen 11 (SPAG11) family were found to play an important role in epididymal innate immunity in addition to their role in sperm maturation. However, the expression of Spag11 transcripts in rodents is not well documented.MethodsComputational analysis was employed to identify novel Spag11 isoforms in the rat. RT-PCR analyses were carried out on RNAs isolated from the male reproductive tract tissues of rat using gene specific primers for Spag11c and Spag11t. The identities of PCR products were confirmed by sequencing. Tissue distribution, developmental expression and androgen regulation of Spag11t and Spag11c were studied using RT-PCR. The antimicrobial activities of recombinant Spag11t and Spag11c were tested against E coli in a colony forming unit assay.ResultsIn this study, we identified two novel Spag11 transcripts, namely, Spag11t and Spag11c derived from the long arm of chromosome 16 in the rat (Rattus norvegicus), using both in silico and molecular biology approaches. Spag11c is expressed in all three regions of the epididymis, in testis and in ovary but is absent from the seminal vesicle. Spag11t expression is confined to the caput and it is not expressed in the testis, seminal vesicle or ovary. Age dependent expression of Spag11t and Spag11c was observed in the epididymides of rats (10–60 day old). Their expression was found to be most abundant in the adult rat (60 day) suggesting roles in mature reproductive function. Further, both Spag11t and Spag11c expression was down regulated in castrated rat epididymides and the expression was maintained in the testosterone replaced castrated rats. SPAG11C is a potent antibacterial agent. SPAG11T also displayed bactericidal capacity although weaker than SPAG11C and SPAG11E.ConclusionThe abundant expression of Spag11t and Spag11c in the male reproductive tract suggests an important role in male reproductive tract immunity. Their expression is developmentally regulated and androgen dependent. Characterization of novel SPAG11 isoforms will contribute to our understanding of the role of epididymal proteins in sperm maturation and innate immunity.

A possible role for progesterone metabolites in human parturition

There has been a recent resurgence of interest in the role of progesterone in the maintenance of human pregnancy and the onset of labour, following recent reports of its use to prevent preterm labour in high-risk patients. One possible mechanism by which progesterone might contribute to uterine quiescence is through the actions of its metabolites. This article provides a brief overview of progesterone in human pregnancy and an outline of progesterone metabolism in the various reproductive tract tissues as well as the evidence for actions of progesterone metabolites in pregnancy.

Identification, cloning and functional characterization of novel beta-defensins in the rat (Rattus norvegicus)

Backgroundbeta-defensins are small cationic peptides that exhibit broad spectrum antimicrobial properties. The majority of beta-defensins identified in humans are predominantly expressed in the male reproductive tract and have roles in non-immunological processes such as sperm maturation and capacitation. Characterization of novel defensins in the male reproductive tract can lead to increased understanding of their dual roles in immunity and sperm maturation.MethodsIn silico rat genomic analyses were used to identify novel beta-defensins related to human defensins 118–123. RNAs isolated from male reproductive tract tissues of rat were reverse transcribed and PCR amplified using gene specific primers for defensins. PCR products were sequenced to confirm their identity. RT-PCR analysis was performed to analyze the tissue distribution, developmental expression and androgen regulation of these defensins. Recombinant defensins were tested against E. coli in a colony forming unit assay to analyze their antimicrobial activities.ResultsNovel beta-defensins, Defb21, Defb24, Defb27, Defb30 and Defb36 were identified in the rat male reproductive tract. Defb30 and Defb36 were the most restricted in expression, whereas the others were expressed in a variety of tissues including the female reproductive tract. Early onset of defensin expression was observed in the epididymides of 10–60 day old rats. Defb21-Defb36 expression in castrated rats was down regulated and maintained at normal levels in testosterone supplemented animals. DEFB24 and DEFB30 proteins showed potent dose and time dependent antibacterial activity.ConclusionRat Defb21, Defb24, Defb27, Defb30 and Defb36 are abundantly expressed in the male reproductive tract where they most likely protect against microbial invasion. They are developmentally regulated and androgen is required for full expression in the adult epididymis.

Differential regulation of neutrophil chemotaxis to IL‐8 and fMLP by GM‐CSF: lack of direct effect of oestradiol

Neutrophils are a normal constituent of the female reproductive tract and their numbers increase in the late secretory phase of the menstrual cycle prior to menses. Several cytokines are produced in female reproductive tract tissue. In particular granulocyte-macrophage colony-stimulating factor (GM-CSF), a potent activator of neutrophils, is secreted in high concentrations by female reproductive tract epithelia. We previously observed that GM-CSF synergizes strongly with interleukin-8 (IL-8) in enhancing chemotaxis of neutrophils. Thus we investigated whether pretreatment of neutrophils with GM-CSF would prime subsequent chemotaxis to IL-8 in the absence of GM-CSF. Surprisingly, a 3-hr pulse of GM-CSF severely diminished chemotaxis to IL-8, whereas N-formyl-methyl-leucyl-phenylalanine (fMLP)-mediated chemotaxis was retained. Conversely, when cells were incubated without GM-CSF they retained IL-8-mediated migration but lost fMLP chemotaxis. These changes in chemotaxis did not correlate with expression of CXCR1, CXCR2 or formyl peptide receptor. However, IL-8-mediated phosphorylation of p44/42 mitogen-activated protein kinase was greatly reduced in neutrophils that no longer migrated to IL-8, and was diminished in cells that no longer migrated to fMLP. Oestradiol, which is reported by some to exert an anti-inflammatory effect on neutrophils, did not change the effects of GM-CSF. These data suggest that neutrophil function may be altered by cytokines such as GM-CSF through modulation of signalling and independently of surface receptor expression.

REGULATION OF SIGNAL TRANSDUCTION PATHWAYS BY ESTROGEN AND PROGESTERONE

The female sex steroid hormones 17beta-estradiol and progesterone mediate their biological effects on development, differentiation, and maintenance of reproductive tract and other target tissues through gene regulation by nuclear steroid receptors that function as ligand-dependent transcription factors. However, not all effects of 17beta-estradiol and progesterone are mediated by direct control of gene expression. These hormones also have rapid stimulatory effects on the activities of a variety of signal transduction molecules and pathways and, in many cases, these effects appear to be initiated from the plasma cell membrane. There is growing evidence that a subpopulation of the conventional nuclear steroid receptor localized at the cell membrane mediates many of the rapid signaling actions of steroid hormones; however, novel membrane receptors unrelated to conventional steroid receptors have also been implicated. This chapter reviews the nature of the receptors that mediate rapid signaling actions of estrogen and progesterone and describes the signaling molecules and pathways involved, the mechanisms by which receptors couple with components of signaling complexes and trigger responses, and the target tissues and cell functions regulated by this mode of steroid hormone action.

T-bet, a key transcriptional factor for Th1 development, is expressed and regulated via Stat family in the epithelial cells of the reproductive tract

T-bet is a key transcriptional factor for Th1 development in T cells. The aim of this study is to understand whether T-bet is expressed in the epithelial cells of the reproductive tract and whether it plays a role in the localized immune environment of the reproductive tract. We examined T-bet expression in immortalized cells derived from endometrium, endocervix, vagina and penile urethera with or without treatment with cytokines and sex hormones. T-bet promoter activity and T-bet function in these cells were also investigated. Cytoplasmic RNA from the above cells was tested for T-bet expression by RT-PCR, real-time PCR, or northern blotting. Nuclear and total cell extracts from these cells were used for western analysis and oligonucleotide precipitation to examine the binding of Stat family to T-bet regulatory region (TRR). Briefly, biotinylated oligonucleotides with the sequence of the IFNγ activation site (GAS), TRR, or mutant TRR were synthesized. Reporter plasmids with TRR or mutant TRR placed upstream of the luciferase gene were transfected into endometrial cells to assess for T-bet promoter activity. The murine T-bet gene was overexpressed in cultured endometrial cells using a construct driven by the CMV promoter to better assess T-bet function. Endogenous T-bet was strongly expressed in vaginal and penile cells, but weakly expressed in endometrial and cervical cells. IFNγ-induced T-bet expression in all epithelial cells studied. Progesterone alters T-bet expression in a biphasic manner in endometrial cells, whereas estrogen suppresses and androgen has no effect on T-bet expression. Stat 1 and 5 bind to the TRR containing a GAS core motif. Mutation of the motif clearly reduced both the binding of the Stat proteins to the TRR and T-bet promoter activity assessed in luciferase assays. IFNγ and IL15 induced the binding of Stat1 and 5, whereas progesterone affects such binding in a biphasic manner. IL15 and IL18 expression was induced after over-expression of T-bet the endometrial cells. After estrogen priming, long-term exposure of endometrial cells to combined progesterone and estrogen activates both T-bet and IL15 expressions (day 8 to 12 after the addition of progesterone). This corresponds to the timing of embryo implantation in vivo. This study demonstrates that T-bet is expressed in the epithelial cells of the reproductive tract, but the expression level is distinct among various reproductive tract tissues. Our data suggest a mechanism by which female sex hormones may promote maintenance of an immune micro-environment favorable to early pregnancy: via the regulation of T-bet expression in endometrial cells.

Estrogen receptor-alpha mediates the detrimental effects of neonatal diethylstilbestrol (DES) exposure in the murine reproductive tract.

It is generally believed that estrogen receptor-dependent and -independent pathways are involved in mediating the developmental effects of the synthetic estrogen, diethylstilbestrol (DES). However, the precise role and extent to which each pathway contributes to the resulting pathologies remains unknown. We have employed the estrogen receptor knockout (ERKO) mice, which lack either estrogen receptor-alpha (alphaERKO or estrogen receptor-beta (betaERKO), to gain insight into the contribution of each ER-dependent pathway in mediating the effects of neonatal DES exposure in the female and male reproductive tract tissues of the mouse. Estrogen receptor-alpha female mice exhibited complete resistance to the chronic effects of neonatal DES exposure that were obvious in exposed wild-type animals, including atrophy and epithelial squamous metaplasia in the uterus; proliferative lesions of the oviduct; and persistent cornification of the vaginal epithelium. DES-mediated reduction in uterine Hoxa10, Hoxa11 and Wnt7a expression that occurs wild-type females during the time of exposure was also absent in alphaERKO females. In the male, alphaERKO mice exhibited complete resistance to the chronic effects of neonatal DES exposure on the prostate, including decreased androgen receptor levels, epithelial hyperplasia, and increased basal cell proliferation. Although ERbeta is highly expressed in the prostate epithelium, DES-exposed betaERKO males exhibited all of the effects of neonatal DES exposure that were observed in similarly exposed wild-type males. Therefore, the lack of DES-effects on gene expression and tissue differentiation in the alphaERKO uterus and prostate provides unequivocal evidence of an obligatory role for ERalpha in mediating the detrimental actions of neonatal DES exposure in the murine reproductive tract.

Bovine papillomavirus type 2 in reproductive tract and gametes of slaughtered bovine females

Papillomaviruses are described selectively infecting epithelial tissues and are associated with many forms of cancer in different species. Considering the widespread dissemination of papillomatosis in livestock, interest is being centred on possible forms of viral transmission and respective mechanisms. In the present study, we report the detection of bovine papillomavirus (BPV) DNA sequences in female reproductive tract tissues, fluids and oocytes from slaughtered bovines not afflicted by cutaneous papillomatosis. BPV-2 DNA sequences were found in ovarian and uterine tissues as well as in oocytes, cumulus cells and uterine flushings. The presence of papillomavirus sequences in reproductive organ tissues and fluids shows that viral infection in organisms can be verified in others tissues, not only in epithelial ones. The present findings alert to the possibility of BPV transmission in embryo transfer programs and assisted fertilization procedures.

Differences in glycosylation and sperm-egg binding inhibition of pregnancy-related glycodelin.

Glycodelin is a glycoprotein produced in many glands, particularly those of reproductive tissues. It appears as different glycoforms in amniotic fluid (glycodelin-A) and seminal plasma (glycodelin-S), but only glycodelin-A inhibits gamete adhesion. In the present study, glycodelin from secretory-phase endometrium, first-trimester pregnancy decidua, and midtrimester amniotic fluid was studied with respect to physicochemical properties, including glycosylation patterns and inhibitory activity of sperm-egg binding. Purified glycodelins from all these sources were similar in isoelectric focusing and in lectin immunoassays using lectins from Wisteria floribunda and Sambucus nigra. Likewise, the glycodelins inhibited sperm-egg binding in a dose-dependent manner, as measured by hemizona-binding assay. However, subtle quantitative physicochemical and biological differences were found between glycodelins from different sources as well as within the same tissue/fluid between different individuals. Differences were most pronounced between endometrial glycodelins from nonpregnancy and first-trimester pregnancy. The glycan structures studied by fast-atom bombardment mass spectrometry of individual amniotic fluid glycodelin-A samples also showed interindividual quantitative differences. In conclusion, glycodelins from different female reproductive tract tissues and amniotic fluid share substantial similarity, allowing all of them to be called glycodelin-A. However, these glycodelins exhibit quantitative physicochemical and functional differences between different sources and individuals.

Mucin gene expression is not regulated by estrogen and/or progesterone in the ocular surface epithelia of mice

Purpose. Dry eye syndrome is prevalent in post-menopausal women, and post-menopausal women secrete less mucus in their reproductive tracts. Using a mouse model, the purpose of this study was to determine if estrogen and/or progesterone regulates Muc4 and Muc5AC gene expression in the ocular surface epithelia, as the hormones do in reproductive tract epithelia. Methods. Adult C57BL/6 mice were ovariectomized, and 19 days later, pellets containing estrogen, progesterone, or a combination were inserted subcutaneously. Ocular surface and reproductive tract tissues were harvested following seven days of hormone treatment. A control group consisted of ovariectomized mice that received no hormone treatment. Real-time reverse transcription-polymerase chain reaction was used to determine the tissue expression levels of mucin mRNA of each treatment group relative to the control. Muc4 mRNA expression levels were determined for the reproductive tract, and both Muc4 and Muc5AC expression levels were determined for the ocular surface epithelia. Muc4 and Muc5AC gene expression in ocular surface and Muc4 in reproductive tract epithelia was demonstrated by In Situ hybridization, and Muc4 and Muc5AC protein was demonstrated in the epithelia of animals in the experimental groups. Results. The mRNA expression levels of Muc4 and Muc5AC and the immunofluorescence localization pattern in the ocular surface epithelia were not significantly different in any hormone treatment group when compared to the control ovariectomized group. By comparison, mice that were administered estrogen had a significant increase of Muc4 mRNA in the reproductive tract epithelia, progesterone given in combination with estrogen antagonized the upregulatory effects of estrogen in the reproductive tract, and the amount of Muc4 mRNA in the reproductive tract of progesterone-treated animals was not different from ovariectomized controls. Immunofluorescence localization of Muc4 in the reproductive tract epithelia of the experimental groups correlated to message levels, with lack of Muc4 protein detected in the control and progesterone groups. Conclusion. In comparison to reproductive tract epithelia, Muc4 and Muc5AC are not hormonally regulated by estrogen or progesterone in the ocular surface epithelia of mice. These data demonstrate that regulation of epithelial mucin genes is tissue specific.

Genetic divergence in cellular resistance to heat shock in cattle: differences between breeds developed in temperate versus hot climates in responses of preimplantation embryos, reproductive tract tissues and lymphocytes to increased culture temperatures

The detrimental effects of heat stress on fertility in cattle are less pronounced in heat-tolerant breeds. Although these genetic differences reflect differences in thermoregulation, cells from heat-tolerant breeds are less adversely compromised by increased temperature (that is, heat shock) than cells from heat-sensitive breeds. Experiments were performed to test the hypothesis that cells and tissues from two thermotolerant breeds (Brahman and Senepol) are better able to survive and function after exposure to increased temperature than cells and tissues from two thermosensitive breeds (Holstein and Angus). Exposure of embryos at>eight-cell stage at day 5 after insemination to heat shock of 41.0 degrees C for 6 h decreased development to the blastocyst stage and the number of cells per embryo. However, the deleterious effect of heat shock on blastocyst formation and the number of cells per embryo was less pronounced for Brahman than for Holstein and Angus breeds. Embryos from Senepol cows had very low development and it was not possible to determine heat shock effects in this breed. In contrast to the sensitivity of embryos to heat shock, there was no effect of a 41.0 degrees C heat shock on [(3)H]leucine incorporation into proteins secreted by oviductal or endometrial explants. Lymphocytes from Brahman and Senepol cows were more resistant to heat-induced apoptosis than lymphocytes from other breeds. Heat shock reduced lymphocyte glutathione content but the magnitude of the decrease was not affected by breed. In conclusion, embryos from Brahman cows are more resistant to heat shock than embryos from Holstein or Angus cows. Genetic differences are also present in thermotolerance for apoptosis response in lymphocytes, with Brahman and Senepol cattle being more resistant to heat shock than Angus and Holstein breeds. It is likely that the evolutionary forces that led to the Brahman and Senepol breeds being adapted to hot climates resulted in the selection of genes controlling resistance to cellular heat shock.

Mouse transgenic for murine oviduct-specific glycoprotein promoter-driven simian virus 40 large T-antigen: tumor formation and its hormonal regulation.

Transgenic mice were generated in which a 2.2-kb segment of the 5'-flanking sequence of the mouse oviduct-specific glycoprotein (OGP) gene was used to drive expression of the simian virus 40 large T antigen (Tag). These mice spontaneously developed tumors in the female reproductive tract. Analysis using reverse transcriptase-polymerase chain reaction showed that the 2.2-kb OGP 5'-flanking region drove Tag mRNA expression in the oviduct, uterus, vagina, and ovary, but not in other tissues. Histological and immunohistochemical analyses revealed that the tumor cells were distributed in the oviduct, endometrium, myometrium, and vagina; and had atypical features, abnormal mitosis, and Tag expression. Ovariectomy suppressed Tag expression, and thereby, blocked tumorigenesis in the transgenic mice. Estradiol administration to ovariectomized transgenic mice led to dramatic hyperplasia of the reproductive tract tissues in association with enhanced Tag expression, both in intensity and distribution. These results demonstrated that a 2.2-kb fragment of the 5'-flanking sequence of the mouse OGP gene was capable of directing the expression of Tag and inducing tumorigenesis in female reproductive tract tissues in an estrogen-dependent manner. Estrogen response elements present in the promoter region were functional in vivo. These transgenic mice represent a unique model, since they develop tumors in the oviducts as well as in other tissues derived from the Mullerian duct.

Altered Levels of Growth-related and Novel Gene Transcripts in Reproductive and Other Tissues of Female Mice Overexpressing Spermidine/Spermine N1-Acetyltransferase (SSAT)

Overexpression of SSAT (polyamine catabolic enzyme) in female mice results in impaired ovarian folliculogenesis and uterine hypoplasia. To identify the molecular basis for this, the gene expression profiles in uterus and ovary and for comparison, liver and kidney, from non-transgenic (NT) and SSAT transgenic (ST) mice were compared. The mRNA abundance for lipoprotein lipase and glyceraldehyde-3-phosphate dehydrogenase was elevated in all four ST (>NT) tissues. The translation initiation factor-3 subunit 5 mRNA, and transcripts related to endogenous murine leukemia provirus (MLV-related) and murine retrovirus-related sequences (MuRRS) were decreased in ST tissues. A novel calmodulin-related mRNA was strongly induced in ST liver and kidney. SSAT overexpression was associated with increased levels of IGF-binding protein-2 (IGFBP-2) in the uterus and ovary, and a reduction in IGFBP-3 mRNA levels in the uterus. Exogenous spermidine and spermine elevated endogenous IGFBP-2 and SSAT mRNA abundance, whereas, putrescine stimulated IGFBP-2 mRNA abundance and transfected IGFBP-2 gene promoter activity in human (Hec-1-A) uterine cells. Sp1 and BTEB1 mRNAs that encode transcription factors for the IGFBP-2 gene also were induced in some ST tissues. The data suggest that SSAT and polyamines are important for the control of molecular pathways underlying reproductive tract tissue growth, phenotype, and function.