Induced ovulation, mating success and embryonic development in the stripe-faced dunnart, Sminthopsis macroura

Induced ovulation resulting in normal embryos is rare in marsupials. In this study natural and induced ovulations were compared in mature Sminthopsis macroura (n = 122). Comparison of maturation of preovulatory oocytes by ovarian histology and examination of oocytes removed from developing follicles in 12 ovaries of 23 animals receiving 0.058 iu equine serum gonadotrophin (eSG) g(-1) with ovaries of 12 animals undergoing natural cycles showed that oocyte maturation was significantly more irregular when it was induced (P < 0.001). Postovulatory stages were examined by estimating the number of eggs ovulated from ovarian histology, and by counting oviduct and uterine contents recovered after ovulation. S. macroura receiving 0.087 iu eSG g(-1) (n = 34), administered as one (n = 17) or two (n = 17) injections, were significantly (P < 0.05) more likely to ovulate (74%), mate (80%) and have conceptuses (66%) than were animals receiving 0.058 iu eSG g(-1) (12, 53 and 0%, respectively) (n = 17), and the values were similar to those in animals (n = 36) undergoing natural cycles (100, 81 and 56%, respectively). Induced ovulation using 0.087 iu eSG g(-1) yielded significantly (P < 0.05) more oocytes per ovary (20.8 +/- 8.5; combined data) than did ovulation in animals undergoing natural cycles (13.7 +/- 3.2) (ANOVA, t test). The responses of animals induced in different phases of the oestrous cycle with 0.087 iu eSG g(-1) were not significantly different (ANOVA) with respect to the number of corpora lutea per ovary, conceptuses per animal or days to ovulation after injection. However, the proportion of females that responded after receiving 0.058 iu eSG g(-1) in the luteal phase was significantly different from that in animals treated with the same dose in the intermediate phase (P < 0.01) and in non-cyclic females treated with 0.058 iu eSG g(-1) (P < 0.02). The main benefits of the treatment were that normal embryos resulted and that 70-78% of non-cyclic animals could be induced to ovulate.     

Significance of serum early pregnancy factor concentrations during pregnancy and embryonic development in Sminthopsis macroura (Spencer) (Marsupialia: Dasyuridae)

Marsupial pregnancy differs from that in eutherians in duration, placentation and hormonal profile so much so that maternal recognition of pregnancy may not occur in polyovular marsupials. However, a comparison of gravid and non-gravid uteri reveals differences indicative of histological and physiological adaptations to pregnancy. In the present study, the hypothesis that embryo-maternal signalling occurs in polyovular marsupials was tested by examining serum from non-pregnant and pregnant Sminthopsis macroura for the presence of early pregnancy factor (EPF), a serum protein secreted by the ovary in response to the presence of a newly fertilized egg in the oviduct. EPF is detectable in the serum of pregnant, but not in non-pregnant, females in all eutherians studied to date. In the present study, EPF was detected in S. macroura serum by the rosette inhibition test during the first 9 days of the 10.7 day gestation period in this marsupial. However, EPF was not detected on day 10, just before parturition, or in non-pregnant or preovulatory animals. Immunohistochemical analysis of ovaries from gravid and non-gravid animals demonstrates that EPF is found in the capillaries, interstitial spaces and secretory cells of the corpus luteum. It is concluded that the spatiotemporal pattern of EPF activity described strongly indicates that maternal recognition of pregnancy in marsupials is mediated, at least in part, by EPF. Because the endocrinological milieu is the same in pregnant and non-pregnant marsupials, the possibility of using marsupials as an experimental system for studying EPF function unconfounded by hormonal effects is presented.     

Induction of ovulation and natural oestrous cycling in the Stripe-faced Dunnart, Sminthopsis macroura

Induced ovulation allows reproduction by otherwise infertile females, and is ideal for the captive breeding of endangered species where the population is aged or breeding is unsuccessful. A predictable time of ovulation after induction has not yet been achieved in polyovular marsupials. Ovulation was induced in Sminthopsis macroura using an initial injection of 20 IU equine serum gonadotrophin (eSG; Day 0), followed on Day 4 by either 20 IU eSG (n = 25) or 0.5 mg porcine luteinizing hormone (n = 26). I.p. hormone injection was given in the morning or early evening, and reproductive status was established prior to induction. Five non-cyclic animals began to cycle naturally following induction and one gave birth to a litter. The time of ovulation after the 1st injection (7.8 +/- 0.9 days) was significantly shorter (P = 0.000) and less variable than the previous study, mimicked the timing of natural cycling, and both natural and induced animals ovulated in the early morning. In vitro oocyte movement through the oviduct, observed for the first time in a marsupial, occurred in pulses. We estimated one group of oocytes could travel the length of the oviduct in 40 min, but it was probably around 4 h. The entire ovulation time (including multiple ovulations) was estimated at 7.5 h. This study has achieved a predictable timing of ovulation after stimulation, and induced noncyclic animals to cycle naturally and give birth, providing a modified methodology for use in captive breeding programs of endangered dasyurid marsupial species with low fecundity.     

Increased milk levels of transforming growth factor-alpha, beta1, and beta2 during Escherichia coli-induced mastitis

Among the gram-negative bacteria that cause mastitis, Escherichia coli are the most prevalent. The innate immune system provides initial protection against E. coli infection by detecting the presence of the foreign pathogens and by mounting an inflammatory response, the latter of which is mediated by cytokines such as IL-1β, IL-8, and tumor necrosis factor (TNF)-α. Although changes in these cytokines during mastitis have been well-described, it is believed that other mediators moderate mammary gland inflammatory responses as well. The growth factors/cytokines transforming growth factor (TGF)-α, TGF-β1, and TGF-β2 are all expressed in the mammary gland and have been implicated in regulating mammary gland development. In other tissues, these growth factors/cytokines have been shown to moderate inflammation. The objective of the current study was to determine whether TGF-α, TGF-β1, and TGF-β2 milk concentrations were altered during the course of E. coli-induced mastitis. The contralateral quarters of 11 midlactating Holstein cows were challenged with either saline or 72 cfu of E. coli, and milk samples were collected. Basal milk levels of TGF-α, TGF-β1, and TGF-β2 were 98.81 ± 22.69 pg/mL, 3.35 ± 0.49 ng/mL, and 22.36 ± 3.78 ng/mL, respectively. Analysis of whey samples derived from E. coli-infected quarters revealed an increase in milk levels of TGF-α within 16 h of challenge, and these increases persisted for an additional 56 h. Elevated TGF-β1 and TGF-β2 milk concentrations were detected in E. coli-infected quarters 32 h after challenge, and these elevations were sustained throughout the study. Because TGF-α, TGF-β1, and TGF-β2 have been implicated in mediating inflammatory processes, their induction during mastitis is consistent with a role for these molecules in mediating mammary gland host innate immune responses to infection.     

Localization of mRNA for vascular endothelial growth factor (VEGF), angiopoietins and their receptors during the peri-implantation period and early pregnancy in marmosets (Callithrix jacchus)

Implantation of a blastocyst into a receptive endometrium is a prerequisite for successful pregnancy. Angiogenesis is a key event in this process but the mechanisms by which localized changes in vascular permeability and angiogenesis occur have yet to be elucidated. Vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEGFR-2 have been implicated as key players in vascular remodelling and placentation. Angiopoietins also appear to have a significant role in regulation of blood vessel growth, maturation and regression. The aim of this study was to describe the molecular regulation of angiogenesis in the first month of pregnancy in marmosets and to address the putative physiological roles for these factors. Uteri were studied at weeks 2, 3 and 4 of pregnancy and compared with late secretory non-pregnant endometrium. Implantation in marmosets occurs at day 11 of pregnancy; hence, these time points were chosen so that the peri-implantation period and very early pregnancy could be studied. mRNAs for VEGF, VEGFR-1 and VEGFR-2, angiopoietin 1, angiopoietin 2 and their receptor Tie-2 were localized and quantified by in situ hybridization. Endothelial cells were identified by CD31 immunocytochemistry. VEGF mRNA was present in all compartments except endothelial cells, and its expression generally increased throughout pregnancy except in upper zone glandular epithelium and luminal epithelium, where a decrease in expression was observed. VEGF receptor mRNAs were found in endothelial cells of the upper zones immediately surrounding glandular epithelium. Angiopoietin 1 mRNA was localized to glandular epithelium of the upper and lower zones throughout pregnancy, and increased in stroma at week 4. Expression of angiopoietin 2 mRNA was localized exclusively to endothelial cells of large luminal vessels and was higher in endometrium from marmosets at week 4 of pregnancy than in endometrium from all other stages. These data provide comprehensive evidence that VEGFR-1 and -2, and angiopoietin 1, angiopoietin 2 and Tie-2 interactions may be involved in the preparation of endometrium for implantation, remodelling of the maternal vasculature and trophoblast invasion during the peri-implantation period in this primate species.     

Expression of vascular endothelial growth factor (VEGF) and its receptors in human endometrium throughout the menstrual cycle and in early pregnancy

Immunohistochemistry for vascular endothelial growth factor (VEGF) and its receptors, fms-like tyrosine kinase (flt-1) and kinase insert domain-containing region (KDR), was performed on human endometrium obtained from patients with normal menstrual cycles, patients given oestrogen and progesterone, and women in early pregnancy. Intense immunostaining of VEGF was observed in both glandular epithelial and stromal cells during the mid-secretory phase; the immunostaining intensity was increased by administration of oestrogen plus progesterone and strong immunostaining was observed in decidual cells of early pregnancy. In addition to the immunostaining in vascular endothelial cells, strong KDR immunostaining was observed in glandular epithelial cells and in decidualized stromal cells induced by administration of oestrogen plus progesterone, whereas flt-1 immunostaining was negligible. Strong immunostaining for flt-1 and KDR was found in both vascular endothelial cells and decidual cells in early pregnancy. Endometrial stromal cells isolated from proliferative phase endometrium were incubated with oestrogen (10(-8) mol l-1) and medroxyprogesterone acetate (MPA; 10(-6) mol l-1) for 18 days to study the regulation of VEGF, flt-1 and KDR in endometrial stromal cells by oestrogen and progesterone. Expression of VEGF and KDR mRNAs was increased significantly by oestrogen and MPA, accompanied by decidualization, whereas flt-1 mRNA expression was not affected. In conclusion, VEGF and its receptors may play important roles in implantation and maintenance of pregnancy.     

Expression of the insulin-like growth factor (IGF) system in the bovine oviduct at oestrus and during early pregnancy

Early mammalian embryo development in vitro can be enhanced by co-culture with oviductal cells and by the addition of insulin-like growth factors (IGFs). This study examined the expression patterns of the oviductal IGF system in cattle in relation to the number of days after oestrus and the presence or absence of embryos. Oviducts were collected from: (i) 66 nulliparous heifers on day 3, day 6 or day 16 after insemination and from (ii) ten non-pregnant, lactating cows on day 0 or day 1 of the oestrous cycle. Oviducts were coiled, frozen whole and sectioned for in situ hybridization. Expression patterns of mRNAs encoding IGF-I, IGF-II, type 1 IGF receptor (IGF-1R), and the IFG binding proteins (IGFBP)-1, -3 and -5 were determined from autoradiographs. Separate measurements were made for the mucosa and muscle layers of the infundibulum, ampulla and isthmus. None of the parameters measured differed between heifers with or without the presence of an embryo. mRNAs encoding IGF-I and IGF-1R were present in the mucosa and muscle of all three oviductal regions, and the highest value of IGF-I mRNA was measured in heifers on day 3. IGF-II mRNA was expressed predominantly in the muscle wall. IGFBP-1 mRNA was not detectable, whereas mRNAs encoding IGFBP-3 and -5 were expressed in both the muscle and mucosa. IGFBP-3 expression was higher in cows on day 0 and day 1 of the oestrous cycle than in heifers on day 3, day 6 and day 16 after insemination. A peak of IGFBP-5 expression was reached on day 6. Locally or systemically produced IGFs, regulated by IGFBPs, may act directly on the embryo or indirectly via modulation of oviductal secretions and muscular activity to influence the success of early embryo development.     

Oestrogen and progesterone receptors in the marmoset endometrium: changes during the ovulatory cycle, early pregnancy and after inhibition of vascular endothelial growth factor, GnRH or ovariectomy

Marmosets are widely used, but detailed studies on localisation of endometrial oestrogen receptors alpha and beta (ER alpha and ER beta ), and the progesterone receptor (PR) are lacking. These receptors were localised and semi-quantitatively analysed throughout the ovulatory cycle, weeks 2, 3 and 4 of pregnancy and after treatment with GnRH antagonist, vascular endothelial growth factor (VEGF) Trap or ovariectomy. The PR in epithelial cells increased markedly between the mid- and late proliferative phases before declining in the mid-secretory phase and pregnancy. PR in stromal cells was present throughout the cycle and levels were maintained in pregnancy. ER alpha was present at the mid-proliferative phase and increased in glands at the late proliferative and early secretory phases, before declining at the late secretory phase and week 4 of pregnancy. Stromal ER alpha showed a similar trend, but decreased earlier, by the mid-secretory phase. ER beta was highly expressed in epithelial cells throughout the cycle and in pregnancy. In stroma, increases in ER beta expression were observed at the late proliferative phase with the staining index decreasing by half as the secretory phase progressed and in pregnancy. GnRH antagonist, VEGF Trap or ovariectomy caused significant reductions in PR and ER beta expression, but not in ER alpha when compared with the late proliferative phase of the normal cycle. Endothelial cells expressed ER beta , but not ER alpha or PR. It is concluded that the steroid receptor profile in the marmoset endometrium is generally similar to the human and should provide a useful model for studies on hormonal manipulation of the endometrium.     

Effect of human growth hormone-releasing factor (1-29)NH2 on growth hormone release and milk production in dairy cows

Twelve cows (209 d in lactation, 642 kg BW) were used in an experiment conducted over four 10-d periods (one preinjection, one injection, and two postinjection). Gelatine (n = 6) or 10 mg of growth hormone-releasing factor in gelatine (n = 6) was injected subcutaneously at 1000 h every day on d 11 to 20. Data were averaged for the last 5 d of each period. During the injection period, milk, fat, and protein yields increased by 3 kg.d-1 (14.3%), .14 kg.d-1 (16.7%), and .12 kg.d-1 (15.4%), respectively. Moreover, milk, fat, and protein yields for the treated cows remained higher than for the control cows until the last postinjection period. Growth hormone response was evaluated from blood samples withdrawn from 2 h prior to 8 h postinjection on d 11, 15, and 20 and on d 40. After growth hormone-releasing factor injection, peaks and area under the curve were 24.5, 30.8, and 47.0 ng.ml-1 and 2475, 3979, and 3741 ng.ml-1.min-1 on d 11, 15, and 20, respectively. On d 40, there was no difference in growth hormone concentrations in blood between control and treated cows. These results demonstrate that 10 d of daily injection of a growth hormone-releasing factor increases milk production by 14.3% (3 kg.d-1) and still induces growth hormone release at the end of the injection period without any sign of refractoriness.     

Expression of fibroblast growth factor-8 and regulation of cognate receptors, fibroblast growth factor receptor-3c and -4, in bovine antral follicles

Paracrine cell signaling is believed to be important for ovarian follicle development, and a role for some members of the fibroblast growth factor (FGF) family has been suggested. In the present study, we tested the hypothesis that FGF-8 and its cognate receptors (FGFR3c and FGFR4) are expressed in bovine antral follicles. RT-PCR was used to analyze bovine Fgf8, Fgfr3c and Fgfr4 mRNA levels in oocytes, and granulosa and theca cells. Fgf8 expression was detected in oocytes and in granulosa and theca cells; this expression pattern differs from that reported in rodents. Granulosa and theca cells, but not oocytes, expressed Fgfr3c, and expression in granulosa cells increased significantly with follicle estradiol content, a major indicator of follicle health. Fgfr4 expression was restricted to theca cells in the follicle, and decreased significantly with increasing follicle size. To investigate the potential regulation of Fgfr3c expression in the bovine granulosa, cells were cultured in serum-free medium with FSH or IGF-I; gene expression was upregulated by FSH but not by IGF-I. The FSH-responsive and developmentally regulated patterns of Fgfr3c mRNA expression suggest that this receptor is a potential mediator of paracrine signaling to granulosa cells during antral follicle growth in cattle.     

Changes in the nitrogen fraction of sainfoin (Onobrychis viciaefolia) during growth

Sainfoin was harvested at four stages of increasing maturity. Samples were analysed for total N, true protein N, non-protein N, nitrate and amino acids. Variations in the concentrations of these nitrogenous fractions throughout the growing period of plant are described.     

Changes in the carbohydrate constituents of yam tuber (dioscorea rotundata,poir) during growth

The amounts of sugars, starch, hemicellulose and cellulose in yam tubers were estimated at different stages of maturity and in yam tuber (“EgbodO”) sections. Maltose, sucrose, glucose and fructose were the free sugars identified in the ethanolic extracts of the yam samples. Sucrose formed the bulk of the total sugars. The “bottom” portion of the “Egbodo” yam tuber was richer in total sugars (4.9%) than either the “middle” (1.8%) or the “head” (2.4%) regions. Starch accounted, respectively, for 60, 77 and 78% of total carbohydrates in the “bottom”, “middle” and “head” regions of the “egbodo” yam tuber. A peak value of 84% starch was obtained 6 months after planting the yam setts. Its decrease to 64% at 8 months has been partly attributed to reduced photosynthetic activity through yellowing and subsequent loss of leaves. The sum of cellulose and hemicellulose, regarded as non-available carbohydrates was less than 7% of the total carbohydrates. The linear fraction, amylose, formed about 17.8% of the starch prepared from whole yam tubers of varying maturities, and from yam tuber sections, the remaining being amylopectin. The observed amylose and amylopectin values did not vary significantly over the period of observation. These amylose and amylopectin fractions had iodine affinities of 17 and 0.1%, respectively.     

Insulin-like growth factor (IGF) system in the oocyte and somatic cells of bovine preantral follicles

Many studies have highlighted the role of the insulin-like growth factor (IGF) system in the control of antral follicular growth. However, much less is known about the involvement of the IGF system in the regulation of preantral follicular development. In an attempt to address this lack of knowledge, the present study describes the spatial and temporal patterns of expression of mRNA encoding components of the IGF system in bovine follicles during preantral stages of development. mRNA was detected by in situ hybridization using frozen sections (14 microm) of bovine ovarian tissue. Serial sections were probed with 35S-labelled bovine riboprobes. Type 1 IGF receptor mRNA was detected in granulosa cells and in the oocyte of preantral follicles; however, in this study, as in previous studies, it was not possible to detect mRNA encoding either IGF-I or -II. IGF binding protein (IGFBP)-2 mRNA was present in granulosa cells and oocytes of preantral follicles, and immunoreactive IGFBP-2 was detected around granulosa cells during this early stage of development. Occasionally, preantral follicles were identified in which there was no expression of IGFBP-2 in granulosa cells or the oocyte. IGFBP-3 mRNA was detected in the oocyte of preantral follicles and in the surrounding stromal tissue. mRNAs encoding IGFBP-2 and -3, and type 1 IGF receptor were first detected in type 2 follicles. In conclusion, although the IGF ligands are not expressed in preantral follicles, mRNAs encoding the type 1 IGF receptor, and IGFBP-2 and -3 were present and showed unique spatial patterns of expression within preantral follicles.     

Studies on growth,structural carbohydrate and phytate in coriander (Coriandrum sativum) during seed development

Coriander (Coriandrum sativum L) is an important spice crop and is used in Indian diets. Physiological characters of plants and biochemical constituents like structural carbohydrate and phytate were studied in developing coriander (var Narnaul and Panth) seeds. Moisture decreased with advancement of seed development. Neutral detergent fibre, acid detergent fibre, hemicellulose, cellulose and lignin levels increased with maturation of seed in both coriander varieties. Significant variations were observed in biochemical constituents between varieties and during different stages of seed development. The presence of phytate did not vary at different stages of seed development. The height of plants varied from 134 to 139 cm with 10 branches. Umbels varied from 89 to 99 with an average of six umbellets. Narnaul seed weight was double that of the variety Panth.     

Some changes in the composition of the fruit of the glasshouse cucumber (Cucumis sativus) during growth, maturation and senescence.

Female cucumber flowers were tagged when fully open and the resultant fruit harvested for growth and chemical analysis at intervals of between 2 and 28 days. The growth curve relating fresh weight and time was sigmoidal approaching a maximum after about 14 days. Similar relationships were obtained for the total content per fruit of sugars, acids and the major nutrients nitrogen, phosphorus and potassium. The percentage dry matter and alcohol-insoluble solids fell rapidly during the first 10 days of growth and then more slowly with increasing fruit age. In contrast, the percentage alcohol-soluble solids changed little during the first 6 days and then decreased progressively. The major sugars, glucose and fructose, present in approximately equal amounts, increased rapidly for the first 6 days and then changed little until the onset of senescence. Traces of sucrose and myoinositol were present at all stages of development. The titratable acidity was extremely low and was closely correlated with the potassium content. On a dry weight basis, total and alcoholsoluble N and P increased slowly during the early stages of growth but more rapidly with increasing maturity. On the other hand, K concentrations tended to decrease.     

Bioeffects of chromium(III) on the growth of Spirulina platensis and its biotransformation

BACKGROUND: In this study the bioeffects of chromium(III) (Cr(III)) on the growth of Spirulina platensis and its biotransformation were investigated. Spirulina platensis was batch cultured in Zarrouk medium containing different concentrations of CrCl₃ · 6H₂O. Total and organic Cr contents in S. platensis were determined by inductively coupled plasma mass spectrometry (ICP‐MS), and Cr speciation was assayed by high‐performance liquid chromatography/ICP‐MS. RESULTS: Results based on the dry weight, growth curve, chlorophyll yield and morphology change of S. platensis indicated that the growth of S. platensis was facilitated by Cr(III) below 4.51 mol L^?1 and inhibited by a higher concentration of Cr(III). It was found that Cr(III) could be accumulated efficiently in S. platensis, with a highest Cr yield of 173.17 g kg^?1 and a highest organic Cr proportion of 50.19 g kg^?1 (96.04% of the total concentration of Cr). No evidence of oxidation of Cr(III) to Cr(VI) in S. platensis cells was found. CONCLUSION: The results of this study suggest that S. platensis could potentially be used as a safe food carrier to accumulate Cr(III) through some metabolic process. Copyright © 2009 Society of Chemical Industry     

Regulation of steroid synthesis and apoptosis by insulin-like growth factor I and insulin-like growth factor binding protein 3 in human corpus luteum during the midluteal phase

The aim of the present study was to investigate the action of insulin-like growth factor I (IGF-I) and insulin-like growth factor-binding protein 3 (IGFBP-3) on steroidogenesis and apoptosis in human corpus luteum during the midluteal phase. Slices from corpora lutea were incubated for 4 h with IGF-I or IGFBP-3. Progesterone, oestradiol, androstenedione and testosterone concentrations were determined by radioimmunoassay; caspase 3 expression was assessed by immunohistochemistry; bcl-2, bax and P(450arom) expression were assessed by RT-PCR; and apoptosis was detected by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling. The results showed that addition of IGF-I stimulated progesterone production (150%, P < 0.01), oestradiol production (65%, P < 0.05) and bcl-2 gene expression (approximately 200%, P < 0.05), but decreased apoptosis (P < 0.05). In contrast, IGFBP-3 reduced steroid production and increased bax gene expression and the percentage of apoptotic cells (P < 0.05). Neither IGF-I nor IGFBP-3 had an effect on P(450arom) expression or on the concentrations of its substrates. However, maximum expression of caspase 3 was detected in corpus luteum during the midluteal phase. In conclusion, these results indicate that IGF-I and IGFBP-3 act as regulatory peptides of the function of the human corpus luteum during the midluteal phase. This action may be direct or mediated by steroid production or by bcl-2-bax expression.