Response to gonadotropin-releasing hormone challenge: Seasonal variation in steroid production in a viviparous lizard, Tiliqua nigrolutea.

The hypothalamic-pituitary-gonadal axis plays a central role in the regulation of gamete maturation, sex steroid production and the stimulation of reproductive behaviours in vertebrates. In seasonal breeders, the timely activation and deactivation of this control system is important to ensure successful reproduction: this process is not well understood in species which breed irregularly. Males of the viviparous blotched blue-tongued lizard, Tiliqua nigrolutea, breed annually, while females display a multiennial cycle. We investigated seasonal variation in hypothalamic-pituitary-gonadal axis responsiveness in both sexes of T. nigrolutea. We measured changes in plasma concentrations of testosterone and estrogen in response to a single intraperitoneal injection of a GnRH agonist, chicken-II LH-RH, at three reproductively distinct times of year. Plasma testosterone concentrations in males were significantly increased during gonadal quiescence, but not initial or final spermatogenesis. There was no estrogen response in males at any time of year. Conversely, in females, there was an increase in plasma testosterone, but not estrogen, concentration, in reproductively quiescent females several months in advance of a successful pregnancy. These results indicate clear variation in HPG axis activity with sex, season and reproductive condition in this seasonally breeding viviparous lizard. This study opens the way for further investigation into the mechanisms by which internal (body condition) and external seasonal cues (temperature and photoperiod) are coordinated to regulate reproduction in irregularly-breeding reptiles.

Endocrine control of embryonic diapause in the Australian sharpnose shark Rhizoprionodon taylori.

The reproductive cycle of the Australian sharpnose shark, Rhizoprionodon taylori, includes a temporary suspension of development at the commencement of embryogenesis termed embryonic diapause. This study investigated levels of 17ß-estradiol (E2), testosterone (T) and progesterone (P4) in plasma samples of mature wild female R. taylori captured throughout the reproductive cycle and correlated them with internal morphological changes. Levels of T were elevated through most of the embryonic diapause period, suggesting a role of this hormone in the maintenance of this condition. Increasing plasma T concentrations from late diapause to early active development were associated with a possible role of androgens in the termination of embryonic diapause. As in other elasmobranchs, a concomitant increase of E2 with ovarian follicle size indicated a direct role of this hormone in regulating vitellogenesis, while a peak in P4 suggested this hormone is associated with preovulation and ovulation. Additionally, significant correlations between photoperiod or water temperature and maximum follicular diameter and hepatosomatic index suggest that these abiotic factors may also play a role triggering and regulating the synchrony and timing of reproductive events.

Yolk contributes steroid to the multidimensional endocrine environment of embryos of Niveoscincus metallicus, a viviparous skink with a moderately complex placenta.

Maternally-derived testosterone (T) and 17-ß-oestradiol (E2) provide epigenetic mechanisms by which mothers can actively influence offspring phenotype. In amniotes, maternal steroids may be derived from yolk or transferred across the placenta according to parity mode. Viviparous reptiles utilise both a yolk and a placenta to support their developing embryos, but it has not yet been confirmed whether yolk is a source of maternal T and E2 in such species. We investigated this question using the viviparous lizard Niveoscincus metallicus as our model species. We measured T and E2 in the yolks during vitellogenesis, immediately post-ovulation and at progressive stages of gestation. Our results confirm that yolk is a substantial source of T and E2 in N. metallicus. Contrary to the pattern seen in many oviparous species, we did not observe a marked decline in yolk concentrations of either T or E2 after the initiation of sexual differentiation in the embryos. Rather, we found no statistically significant decline in yolk concentrations of both T and E2 post-ovulation. In viviparous reptiles that utilise both yolk and placenta to nourish their embryos, yolk likely plays an important role in these dynamics but that role is not yet clear. Further research is warranted to understand the importance of yolk steroids in the endocrine environment of the developing viviparous reptile.

Neurotransplantation of stem cells genetically modified to express human dopamine transporter reduces alcohol consumption.

INTRODUCTION: Regulated neurotransmitter actions in the mammalian central nervous system determine brain function and control peripheral organs and behavior. Although drug-seeking behaviors, including alcohol consumption, depend on central neurotransmission, modification of neurotransmitter actions in specific brain nuclei remains challenging. Herein, we report a novel approach for neurotransmission modification in vivo by transplantation of stem cells engineered to take up the neurotransmitter dopamine (DA) efficiently through the action of the human dopamine transporter (hDAT). As a functional test in mice, we used voluntary alcohol consumption, which is known to release DA in nucleus accumbens (NAC), an event hypothesized to help maintain drug-seeking behavior. We reasoned that reducing extracellular DA levels, by engrafting into NAC DA-sequestering stem cells expressing hDAT, would alter alcohol intake. METHODS: We have generated a neural stem cell line stably expressing the hDAT. Uptake kinetics of DA were determined to select a clone for transplantation. These genetically modified stem cells (or cells transfected with a construct lacking the hDAT sequence) were transplanted bilaterally into the NAC of wild-type mice trained to consume 10% alcohol in a two-bottle free-choice test for alcohol consumption. Alcohol intake was then ascertained for 1 week after transplantation, and brain sections through the NAC were examined for surviving grafted cells. RESULTS: Modified stem cells expressed hDAT and uptaken DA selectively via hDAT. Mice accustomed to drinking 10% ethanol by free choice reduced their alcohol consumption after being transplanted with hDAT-expressing stem cells. By contrast, control stem cells lacked that effect. Histologic examination revealed surviving stem cells in the NAC of all engrafted brains. CONCLUSIONS: Our findings represent proof of principle suggesting that genetically engineered stem cells can be useful for exploring the role of neurotransmitters (or other signaling molecules) in alcohol consumption and potentially in other aspects of brain function.

"Are you at risk for hereditary breast cancer?": development of a personal risk assessment tool for hereditary breast and ovarian cancer.

Identification of risk for the hereditary breast and ovarian cancer syndrome (HBOC) is important, as research has demonstrated the benefits of risk-reducing interventions for women with or at risk for this disorder. Knowledge among women regarding risk factors for hereditary breast cancer and the existence of cancer genetics services appears limited. The goal of this project was to develop a tool to broaden women's awareness regarding their potential risk for HBOC. A formal instructional design process was used to develop a brochure to facilitate recognition of HBOC risk among women attending a no-cost breast and cervical cancer screening clinic. Brochure development was guided by gathering feedback from potential users early and often. The resulting brochure included four parts: (1) a brief description of the impact of hereditary breast cancer risk on one's health; (2) a personal and family history collection table; (3) a series of questions enabling the user to self-assess HBOC risk; (4) a list of resources for women at risk for HBOC. User feedback indicated that the brochure was easy to use. The project demonstrated that women can self-evaluate their risk for HBOC. Future work will evaluate this tool among a broader population of women.

Uterine epithelial changes during placentation in the viviparous skink Eulamprus tympanum.

We used scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to describe the complete ontogeny of simple placentation and the development of both the yolk sac placentae and chorioallantoic placentae from nonreproductive through postparturition phases in the maternal uterine epithelium of the Australian skink, Eulamprus tympanum. We chose E. tympanum, a species with a simple, noninvasive placenta, and which we know, has little net nutrient uptake during gestation to develop hypotheses about placental function and to identify any difference between the oviparous and viviparous conditions. Placental differentiation into the chorioallantoic placenta and yolk sac placenta occurs from embryonic Stage 29; both placentae are simple structures without specialized features for materno/fetal connection. The uterine epithelial cells are not squamous as previously described by Claire Weekes, but are columnar, becoming increasingly attenuated because of the pressure of the impinging underlying capillaries as gestation progresses. When the females are nonreproductive, the luminal uterine surface is flat and the microvillous cells that contain electron-dense vesicles partly obscure the ciliated cells. As vitellogenesis progresses, the microvillous cells are less hypertrophied than in nonreproductive females. After ovulation and fertilization, there is no regional differentiation of the uterine epithelium around the circumference of the egg. The first differentiation, associated with the chorioallantoic placentae and yolk sac placentae, occurs at embryonic Stage 29 and continues through to Stage 39. As gestation proceeds, the uterine chorioallantoic placenta forms ridges, the microvillous cells become less hypertrophied, ciliated cells are less abundant, the underlying blood vessels increase in size, and the gland openings at the uterine surface are more apparent. In contrast, the yolk sac placenta has no particular folding with cells having a random orientation and where the microvillous cells remain hypertrophied throughout gestation. However, the ciliated cells become less abundant as gestation proceeds, as also seen in the chorioallantoic placenta. Secretory vesicles are visible in the uterine lumen. All placental differentiation and cell detail is lost at Stage 40, and the uterine structure has returned to the nonreproductive condition within 2 weeks. Circulating progesterone concentrations begin to rise during late vitellogenesis, peak at embryonic Stages 28-30, and decline after Stage 35 in the later stages of gestation. The coincidence between the time of oviposition and placental differentiation demonstrates a similarity during gestation in the uterus between oviparous and simple placental viviparous squamates.

In vitro steroid production by adrenals and kidney-gonads from embryonic southern snow skinks (Niveoscincus microlepidotus): implications for the control of the timing of parturition?

In some mammalian species, hormones produced by the embryo(s) at the completion of development are involved in the cascade of events that result in parturition. Our overall aim was to determine whether a similar mechanism exists in viviparous reptiles. The alpine skink Niveoscincus microlepidotus provides a useful model for studies of gestation and parturition in viviparous reptiles as the completion of embryonic development and parturition are temporally separated; ovulation occurs in spring, embryonic development is completed by autumn, but parturition does not occur until the following spring. In this study, we determined how in vitro steroid hormone production by embryonic adrenals (progesterone, corticosterone, and testosterone) and mesonephric kidney-gonads (oestradiol and testosterone) varied during the later stages of gestation. We hypothesised that embryonic adrenals and kidney-gonads were capable of producing steroid hormones in vitro, and that the pattern of production would change as parturition approached, would be influenced by temperature, and would increase in the presence of corticotropin (ACTH) or pregnenolone, respectively. Embryonic adrenals and kidney-gonads were incubated with or without ACTH or the steroid precursor pregnenolone, respectively. Tissues were incubated for 3 h at either 16 or 24 degrees C (preferred body temperature of pregnant N. microlepidotus). Incubation medium was analysed for steroid hormones using radioimmunoassay. Low levels of progesterone were produced in vitro during the later stages of gestation when embryonic adrenals were incubated with ACTH. In vitro corticosterone production by embryonic adrenals also occurred, with greater production occurring when tissues were incubated at 24 degrees C. Testosterone was produced in vitro by both adrenals and kidney-gonads in March (late autumn when embryonic development was complete, but prior to parturition the following spring), with greater production at 16 degrees C. This peak in testosterone production coincided with differentiation and continuing growth of the hemipenes. Low levels of oestradiol were produced in vitro by embryonic kidney-gonads in March. These results provide some support for the hypothesis that corticosterone production by the embryonic adrenal may have a role in determining the timing of parturition in a viviparous reptilian species. Further research is needed to differentiate the activity of various tissues and steroid hormones in control of embryonic development, sexual differentiation, and the potential regulation of gestation and parturition in N. microlepidotus and other viviparous reptiles.

Seasonal variations in reproductive hormones in free-ranging echidnas (Tachyglossus aculeatus): interaction between reproduction and hibernation.

We measured the reproductive steroids testosterone and progesterone in free-ranging adult echidnas over several years. For months other than June-August, the mean progesterone concentration was 0.18+/-0.12 ng ml(-1) (n=14), and all blood samples taken from active female echidnas in June-August had progesterone concentrations above 0.5 ng ml(-1). The highest progesterone value measured was 13.4 ng ml(-1) in a pregnant female several days before egg-laying. For months other than June-August the mean testosterone concentration was 0.09+/-0.05 ng ml(-1) (n=13). During June-August all active sexually mature males had testosterone concentrations in excess of 0.2 ng ml(-1) and were found in mating groups at some time during this period. The highest plasma testosterone concentration measured was 4.62 ng ml(-1).

Adrenal and white cell count responses to chronic stress in gestating and postpartum females of the viviparous skink Egernia whitii (Scincidae).

This study investigates the relationships between plasma corticosterone concentrations and white cell counts in captive females of the viviparous lizard Egernia whitii during two phases of the reproductive cycle. Gestating and postpartum females were captured in the field and held in the laboratory for 4 weeks. Plasma corticosterone and progesterone concentrations and white blood cell counts were examined in blood samples taken at capture and after 24 h, 1 week, and 4 weeks in captivity. At 24 h after capture, plasma corticosterone concentrations in both groups had increased significantly compared with initial values but then returned to initial concentrations after 1 week in captivity and remained low in the 4 week samples. Plasma progesterone concentrations remained elevated in the gestating females until the week 4 sample, just prior to parturition. The hormone data suggest that capture and captivity did not represent a significant long-term stressor to these animals. The increase in plasma corticosterone concentration was associated with heterophilia in the differential leucocyte count in both groups of females. Lymphocyte numbers decreased only in gestating females, suggesting that reproductive status may influence the interaction between adrenal activity and immune function.

Patterns of peripheral steroid metabolism vary with sex, season, and tissue type in blotched blue-tongued lizards (Tiliqua nigrolutea).

We examined sexual and seasonal variation in the ability of reproductively relevant tissues (liver, skin, adrenal gland, cloaca, kidney, renal sexual segment, epididymis, oviduct, muscle, testis, and ovary) to metabolise a primary steroid [testosterone (T) or estradiol (E2)] in the scincid lizard, Tiliqua nigrolutea. We observed considerable variation between sexes and across seasons in the patterns of conjugation and derivatisation of the primary steroids by these tissues. All tissues demonstrated the ability to conjugate the relevant primary steroid. Other general trends included increased conjugation by all tissues of gestating females, reduced metabolism of E2 by female tissues during late vitellogenesis, and reduced metabolism of T by males during early spermatogenesis. 5alpha-Dihydrotestosterone was the most commonly detected derivative in males, and production varied with season and tissue type. We suggest that seasonal variation in the ability of reproductively relevant tissues may be important in the physiological regulation of reproduction in this species.

Information needs about hereditary breast cancer among women with early-onset breast cancer.

To explore hereditary breast cancer (HBC) information needs of early-onset breast cancer survivors, two questionnaires were mailed to women diagnosed at age <50 years. The Family History Questionnaire, sent to women treated at 34 Virginia hospitals, was designed to identify women with suspected HBC versus women with presumed sporadic breast cancer (SBC). Among 314 respondents, 137 reported personal/family histories suggestive of HBC. A total of 287 (87%) participants responded to the subsequent Knowledge, Attitudes, and Beliefs Questionnaire, which assessed HBC knowledge, attitudes, and beliefs, including HBC information needs, sources, and perceived value. Fifty-two percent of women reported seeking any HBC information. Women with presumed SBC were as likely to seek information as those with suspected HBC. Women with daughters were more likely to seek information. Many (71%) women reported finding the information sought, including information about their children's and relatives' risks, DNA testing, treatment differences for HBC, and genetic counseling.

In vitro progesterone production by maternal and embryonic tissues during gestation in the southern snow skink (Niveoscincus microlepidotus).

The southern snow skink, Niveoscincus microlepidotus, has a protracted gestation, lasting approximately one year. Ovulation occurs in spring (November) and embryonic development is completed by early autumn (March); however, birth does not occur until the following spring. Previous studies have shown that plasma progesterone concentration peaks in preovulatory females (spring, October), remains high during early gestation, and decreases to basal by autumn. In vitro progesterone production by corpora lutea, non-luteal ovary, anterior oviduct, placental tissues, muscle, and embryonic adrenal-gonads from N. microlepidotus was assessed throughout gestation. Tissues were incubated with or without the precursor pregnenolone for 3h at 24 degrees C; the resulting media were analysed for progesterone using radioimmunoassay. In vitro progesterone production by corpora lutea in media only was high during early gestation, dropping to basal by autumn. Maternal adrenal glands produced progesterone in vitro in media only throughout gestation; however, the pattern of production did not correlate with plasma concentrations and may represent steroid that is normally converted to corticosterone. Non-luteal ovary, anterior oviduct, placental tissues, muscle, and embryonic adrenal-gonads produced minimal progesterone in media only, but were able to convert pregnenolone to progesterone; this suggests steroid metabolic capability within these tissues. Further research is needed to address the possible endocrine role(s) of placental and embryonic tissues during gestation in viviparous squamates.

Breast cancer survivors' attitudes about communication of breast cancer risk to their children.

Perceptions of breast cancer survivors regarding educating their children about hereditary breast cancer risk are underexplored. This study examined attitudes of early-onset breast cancer survivors concerning whether, at what age, and how their children should be educated about potential risk for hereditary breast cancer. Women with breast cancer diagnosed < age 50 years between 1994 and 1997 were recruited from 34 Virginia hospitals. Participants responded to two surveys. The Family History Questionnaire permitted participant classification into two groups based on risk for hereditary disease, as determined by personal and family history. The Knowledge, Attitudes and Beliefs Questionnaire addressed participants' concerns regarding their children's breast cancer risk, perceptions of their children's concerns about their own risk, and whether, at what age, and from what source their children should be informed about their own risk. Among 267 participants, the average age was 47.5 years; 90% were Caucasian, 7% were African American; 52% had suspected hereditary breast cancer. Of participants with children, 81% indicated concern about their children's breast cancer risk; 55% reported that their children had expressed concern about their own risk. Seventy-one percent of participants believed the age for informing children about hereditary breast cancer risk to be before 18 years. Eighty-four percent cited the child's parents and 65% cited health professionals as a preferred information source. No differences in responses between participants with suspected hereditary breast cancer versus presumed sporadic breast cancer were noted. These data establish the need for educational resources regarding hereditary breast cancer for children of early-onset breast cancer survivors.

A possible alternative to 17beta-estradiol in a viviparous lizard, Tiliqua nigrolutea.

We have detected an unusual polar steroid as a major end product of gonadal steroid biosynthesis in male and female blotched blue-tongued lizards, Tiliqua nigrolutea. In an investigation of the steroidogenic pathways in blue-tongued lizards, we incubated gonads of both male and female lizards with tritiated pregnenolone and identified the products of steroid biosynthesis using thin layer chromatography and high performance liquid chromatography with radiometric detection: gonads from both reproductively active and quiescent lizards were examined. The proportion of total radioactive substrate converted to the unknown polar steroid was greater in individuals with hypertrophied gonads (males (N=4) 28.1%; females (N=4) 45.3%) than in those with regressed gonads (males (N=4) 5.9%; females (N=4) 33.3%). Properties of the unknown steroid were identical across all incubations. Incubation time-course information, thin layer and high performance liquid chromatography properties of the polar steroid are presented. The steroid is more polar than 17beta-estradiol, which was not detected, but less polar than all tri-hydroxylated estrogens to which it was compared. The possibility of an alternative estrogen to 17beta-estradiol in some reptiles is discussed.

Interrelationships among plasma progesterone concentrations, luteal anatomy and function, and placental ontogeny during gestation in a viviparous lizard (Niveoscincus metallicus: Scincidae).

Plasma progesterone concentrations were measured at six stages of gestation in the viviparous lizard Niveoscincus metallicus. Anatomical and functional parameters of luteal activity were also investigated. The diameter of the corpus luteum (CL) decreased gradually though gestation, as did the diameter of the luteal cells. Major degenerative changes were observed in CLs post-partum. Plasma progesterone concentrations were basal both prior to, and just after, ovulation; a rapid increase occurred in early gestation. Plasma progesterone concentrations remained elevated until late gestation, but fell some 2 weeks before parturition. In vitro production of progesterone was greater in CLs in mid- than in late-gestation, and the addition of prostaglandin F(2alpha) to the incubation medium had no effect on progesterone production. Non-luteal ovarian tissue and adrenals produced progesterone, but at approximately one-tenth the rate of production by CLs. Temporal correlations between the plasma progesterone profile and stages of placental development were also assessed. The rise in plasma progesterone concentrations occurs before differentiation of the chorioallantoic placenta, but progesterone is still high when it degenerates. We conclude that the CLs are the major source of gestational progesterone in N. metallicus.