hCG Treatment Research Articles

Development of in vitro culture method for early stage zebrafish (Danio rerio) ovarian follicles for use in cryopreservation studies

There have been no reported methods for in vitro growth of early stage ovarian follicles for fish and their cryopreservation is still under investigation. If cryopreservation of early stage ovarian follicles can be achieved, in vitro procedures for ovarian follicle culture, development, ovulation and fertilisation after cryopreservation would be needed. The aim of the present study was to develop an in vitro culture method for early stage zebrafish ovarian follicles for use after their cryopreservation. Procedures for in vitro culture of stage I (primary growth) and stage II (cortical alveolus) ovarian follicles were developed. The effects of concentration of L-15 medium, pH and the concentration of human chorionic gonadotropin (hCG) and activin A were studied. The results demonstrated that early stage zebrafish ovarian follicles can be cultured in vitro for 24 h, stage I and II ovarian follicles can grow to the sizes of early stage II and stage III ovarian follicles after hCG treatment. The method developed here is effective for assessing early stage zebrafish ovarian follicles growth competence in vitro. The results from the present study indicated that in vitro culture is the most reliable method for assessing ovarian follicle viability when compared with vital dye staining methods.

Temporal and periovulatory changes in ovarian catecholamines in the catfish Heteropneustes fossilis

A high performance liquid chromatography-fluorescence detection method was employed to demonstrate temporal and periovulatory changes in ovarian catecholamines in the catfish Heteropneustes fossilis. Tyrosine, l-DOPA, dopamine (DA), norepinephrine (NE) and epinephrine showed significant seasonal and diurnal changes during the reproductive cycle. A high concentration of tyrosine was detected, the values increased in day and decreased in night during recrudescence from preparatory to spawning phases. Similarly, l-DOPA increased during the breeding phase giving the day value concentration peak in the spawning phase and the night value peak in the postspawning phase. DA activity or turnover index (calculated as a ratio of DA to 3,4-dihydroxyphenylacetic acid, DOPAC) showed a bimodal pattern with the major activity peak in the postspawning phase and the minor one in the prespawning phase, the spawning phase registered the lowest activity. NE activity or turnover index (ratio of NE to normetanephrine, NME) increased during the recrudescent phase to give the peak in the spawning phase and decreased in the quiescent phase. Epinephrine elicited an inverse relationship in the day–night pattern, the day values increased to the peak in the spawning phase. All the study correlates showed significant periovulatory changes after hCG treatment. DA activity dropped to the nadir at 8 h but recovered at 16 and 24 h. NE activity showed only a marginal decrease up to 16 h but decreased drastically at 24 h. Epinephrine levels remained unchanged but only to increase at 24 h. The seasonal patterns and periovulatory changes strongly point to important functions for catecholamines in the ovary. The occurrence of tyramine (an invertebrate neurotransmitter) suggests the presence of alternate catecholamine pathway in fish ovary.

Intra-uterine insemination with low numbers of frozen–thawed boar spermatozoa in spontaneous and induced ovulating sows under field conditions

The present study was conducted to evaluate non-return rate (NR), farrowing rate (FR), and number of total pigs born/litter (TB) of weaned sows after intra-uterine insemination (IUI) using low numbers of frozen–thawed (FT) spermatozoa. Semen from 6 boars was cryopreserved individually in a 0.5-ml straw, at a concentration of 1 × 10 9 spermatozoa/ml. A total of 40 multiparous sows with weaning-to-estrus interval of 3 to 7 days were included. The sows were detected for standing estrus twice daily and randomly assigned to two groups: I) spontaneous ovulation ( n = 20) and II) induced ovulation ( n = 20) which the sows were given 750 IU human chorionic gonadotrophin (hCG) i.m. immediately at estrus detection. Ovulation was determined every 12 h using transrectal ultrasonography. FT semen containing 1 × 10 9 motile spermatozoa/dose was used to IUI. In group I, the sows were inseminated at 24 h after the detection of estrus and repeated every 12 h until ovulation. In group II, the sows were inseminated at 36, 42 and/or 48 h after hCG treatment. The results showed that the interval from standing estrus to ovulation (EOI) differed significantly between group I (40.2 h) and group II (35.6 h; P = 0.01). Variation of EOI among sows within each group seemed to be lower in group II (4.5 h SD) than in group I (5.5 h SD; P = 0.5). The number of IUI per sow was 2.9 ± 0.6 times in group I and was 2.4 ± 0.5 times in group II. There were no significant differences ( P > 0.05) in the NR (80 vs 85%), FR (60 vs 65%) and the TB (8.0 ± 2.8 vs 9.4 ± 3.7 piglets/litter) between the groups. These results indicated that multiple doses of IUI with a low number of FT boar spermatozoa provided a fairly good NR, and reasonable FR and TB both in spontaneous and induced ovulating sows. The number of inseminations required for attaining acceptable fertility tended to be lower in the weaned sows with induced ovulation.

Effect of gonadotropin treatment on estrus, ovulation, and litter size in weaned and anestrous sows1

In the first of 2 experiments, we evaluated the effects on anestrous sows of pretreatment with FSH to stimulate the growth of small follicles, followed by eCG to stimulate the growth of medium follicles, estrus, and ovulation. In Exp. 2, we examined the effect of sows receiving 400 IU of eCG plus 200 IU of hCG (PG 600, Intervet/Schering Plough Animal Health, Boxmeer, the Netherlands) at weaning and then different doses and timing of supplemental hCG. In Exp. 1, a total of 87 multiparous Hypor sows deemed anestrus 7 d after weaning were assigned to intramuscular (i.m.) injection of 1) PG 600, 2) eCG (600 IU), 3) pretreatment with 87.5 IU of FSH on d 7 and 8 plus eCG on d 9, or were 4) noninjected controls. Sows had daily boar contact for 15 d after weaning for estrus detection. Blood samples were obtained on d 9 and 19 and assayed for progesterone to determine ovulation status. The weaning-to-estrus interval, number of sows in estrus and ovulating, farrowing rate, and litter size were not different (P > 0.1) in treated groups compared with controls. In Exp. 2, a total of 247 Hypor sows were assigned at weaning by parity (1 and 2 or > or = 3) to receive 1) an i.m. injection of PG 600, 2) PG 600 supplemented with 100 IU of hCG injected either concurrently or after 24 h, 3) 200 IU of hCG after 24 h, or 4) no injection (controls). Sows were exposed to boars daily for 7 d. After treatment of parity 1 and 2 sows, all gonadotropin-treated groups had an increased (P < 0.05) number of sows in estrus compared with the control group; weaning-to-estrus interval, farrowing rates, and litter size were unaffected (P > 0.1). After treatment of parity > or = 3 sows, there was no treatment effect on the estrous response and weaning-to-estrus interval; compared with control and PG 600-treated sows, farrowing rate was decreased (P < 0.05) for sows receiving 200 IU of hCG after 24 h. There was no effect (P > 0.1) of treatment on litter size. We conclude that gonadotropins can be used to increase estrus response in weaned sows, but that hCG treatment subsequent to PG 600 may be detrimental to sow fertility in parity > or = 3 sows.

Cytochrome P450 aromatases: Impact on gonadal development, recrudescence and effect of hCG in the catfish, Clarias gariepinus

Present study analyzed the importance of two forms of aromatases during ovarian development and recrudescence of north African/air-breathing catfish. We cloned both CYP19A1 (1941 bp; ovarian form) and CYP19A2 (1786 bp; brain form), which showed 47% homology between the two forms. Characterization of encoded proteins in non-steroidogenic COS-7 cells illustrated that both isoforms efficiently catalyzed the aromatization reaction by producing estradiol-17β (E 2) from testosterone. Tissue distribution pattern revealed preferential expression of CYP19A2 in brain while CYP19A1 predominated in ovary with trace amounts detected in other tissues including brain. Relative real-time PCR analysis revealed high transcript levels of both isoforms in the prespawning phase of ovarian cycle, which is in accordance with serum E 2 level. Aromatase activity in brain was comparatively lower than ovary, indicating the predominant requirement of aromatase in ovary. Ontogeny studies displayed sexual dimorphism, with early expression of CYP19A1 and CYP19A2 in ovary and brain, respectively. Phase-dependent rise of expression and enzyme activity of aromatase after hCG treatment revealed the stimulatory role of gonadotropin during preparatory and prespawning phases, preferentially to promote vitellogenesis. Lack of influence of hCG treatment during spawning phase endorses it further. A good correlation of expression, enzyme activity and serum E 2 levels suggests a crucial role of CYP19A1 during ovarian differentiation and ovarian cycle of catfish. Likewise, CYP19A2 might also be involved in these processes either indirectly or directly.

Human chorionic gonadotropin: A hormone with immunological and angiogenic properties

The success of implantation depends on a receptive endometrium, a normal blastocyst and synchronized cross-talk at the maternal-fetal interface. The progression of pregnancy then requires immunological tolerance which allows conceptus survival. A cascade of cytokines mediates this dialogue and is crucial in the cross-talk between the immune and endocrine systems. The first known human embryo-derived signal is chorionic gonadotropin (hCG) by which the embryo profoundly influences immunological tolerance and angiogenesis at the maternal-fetal interface. hCG levels coincide with the development of trophoblast tolerance. Indeed, it increases the number of uterine natural killer cells that play a key role in the establishment of pregnancy. hCG also intervenes in the development of local immune tolerance through the cellular system of apoptosis via Fas/Fas-Ligand. It modulates the Th1/Th2 balance and acts on complement C3 and C4A/B factors modulating decidual immunity. The transient tolerance evident during gestation is at least partially achieved via the presence of regulatory T cells which are attracted by hCG at the fetal-maternal interface. Finally, hCG treatment of activated dendritic cells results in an up-regulation of MHC class II, IL-10 and IDO expression, reducing the ability to stimulate T cell proliferation. Successful implantation requires an extensive endometrial angiogenesis in the implantation site. Recent data demonstrate angiogenic effects of hCG via its interaction with endometrial and endothelial LH/hCG receptors. Our review focuses on these functions of hCG, giving new insight into the endocrine-immune dialogue that exists between the conceptus and immune cells within the receptive endometrium at the time of implantation.

Transforming growth factor-β (TGFβ) and its signaling components in peri-ovulatory pig follicles

The present studies investigated the hypothesis that TGFβ plays a role in mediating LH/hCG-induced maturation, ovulation and/or luteinization of follicles in the pig. In Experiment 1, the temporal and spatial gene expression patterns of TGFβ signaling components were examined in pig follicles which had been induced to ovulate and luteinize in vivo by hCG treatment, or by the LH-surge. Pre-pubertal pigs were injected with PG-600 followed by hCG, and ovaries were collected surgically at 0, 1, 12, 24 and 48 h post-hCG. Post-ovulatory follicles were also collected from cycling gilts on Day 4 (D4) of the estrous cycle. Pre- and post-ovulatory follicles were used for the measurement of mRNA (PCR) and protein (Western blots) abundance and for protein localization by immunohistochemistry (IHC). Steady state amounts of mRNA for TGFβ3 and TGFβR2 were increased ( P < 0.05, as compared to 0 h) at 12 h and on D4, respectively, while TGFβ2 protein showed a tendency to increase on D4. TGFβ signaling components did not change significantly. By IHC, the localization of TGFβ components was as follows: pre-ovulatory follicles; TGFβ1 – granulosa cells (GC), TGFβ2 – theca cells (TC), TGFβR1 and 2 – GC and TC: post-ovulatory follicles; TGFβ1 and 2 and TGFβR1 and 2 – luteinizing TC and GC. In Experiment 2, TGFβ1 (1–100 ng/ml) alone had no significant effect on progesterone (P4) secretion by pig GC in culture. Furthermore, while LH + IGF-1 (positive control) stimulated P4 ∼10-fold, TGFβ at 10 and 100 ng/ml added in combination with LH + IGF-1, had no effect on P4 accumulation. In conclusion, data from the present study on temporal and spatial patterns of expression of the TGFβ-system suggest that TGFβ may play a role in the overall process of luteinization, but it appears not to influence steroidogenesis in luteinizing pig follicles.

Repeated administration of hCG on follicular and luteal characteristics and serum progesterone concentrations in eCG-superovulated does

The objective of this study was to evaluate the effect of repeated administration of hCG on the follicular and luteal charactristics and serum progesterone concentrations in eCG-superovulated Merghoz does. The estrous cycles of 40 adult does were synchronized following a 12-day treatment with intravaginal sponges (Chronogest ®), with 1000 IU eCG injected 2 days prior to sponge removal. On the day of sponge removal (day 0), the females were randomly assigned to 4 treatments. The first group (h1; n = 10), received an i.m. hCG injection (500 IU; Pregnyl ®) on the day 0. The second group (h2; n = 10), received an i.m. hCG (500 IU) injection on days 0 and 1, while the third group (h3; n = 10) received i.m. hCG (500 IU) on days 0–2. Does in the control group ( n = 10) did not receive any hCG treatment. Blood samples were taken on days 0, 1, 2, 5 and 8 and analysed for serum progesterone (P4) concentrations. Premature luteal regression was considered to have occurred if the P4 concentrations declined to less than 1 ng/mL by day 8 following sponge removal. On day 8 goats were slaughtered and ovaries recovered for evaluation. In each case the CL's were counted and classified according to their appearance – as normal or as regressing. According to the serum P4 concentration on day 8, 42.8% (3/7), 0% (0/8), 0% (0/7) and 0% (0/6) of the goats underwent premature luteal regression in the control, h1, h2 and h3 groups, respectively. The control group was the only group in which there was a significant increase in the number of regressing CL's on day 8 (5.1 ± 1.5; P < 0.05). The characteristics of the CL's, including the mean diameter and weight was greater ( P < 0.05) in the h2 and h3 groups, compared to the control group (10.9 ± 0.7, 11.0 ± 0.9 vs. 7.9 ± 1.1 mm and 640.0 ± 101.4, 691.7 ± 111.7 vs. 294.3 ± 143.7 mg, respectively). No differences ( P < 0.05) in the number of large follicles present on the ovaries were recorded on day 8 between the groups. However, the diameter of the large follicles present on the ovaries on day 8 was greater ( P < 0.05) in goats in the h2 and the h3 groups, compared to the control (15.3 ± 0.6 and 12.9 ± 0.3 vs. 8.6 ± 1.7 mm, respectively; P < 0.05). The serum P4 concentrations were higher in the h2 and the h3 groups than in the control group on days 2 and 8. The serum P4 concentrations on day 8 also recorded a positive correlation with the normal CL's, CL diameter and CL weight ( P < 0.001). These results indicate that the repeated administration of hCG post-sponge removal presumably prevents premature luteal regression and improves luteal function in goats after eCG-superovulatory treatment. However, neither the number of persistent follicles nor the diameter of the persistent follicles was reduced.

The management and outcome of women with post-hydatidiform mole ‘low-risk’ gestational trophoblastic neoplasia, but hCG levels in excess of 100 000 IU l−1

Background:Gestational trophoblastic neoplasia (GTN) after a hydatidiform mole is either treated with single- or multi-agent chemotherapy determined by a multifactorial scoring system. Women with human chorionic gonadotrophin (hCG) levels >100 000 IU l−1 can remain within the low-risk/single-agent category and usually choose one drug therapy. Here we compare the success and duration of single- vs multi-agent chemotherapy in this patient group.Methods:Between 1980 and 2008, 65 women had a pre-treatment hCG >100 000 IU l−1 and were low risk. The initial hCG level, treatment regimens, changes and duration and overall survival were recorded.Results:Of 37 patients starting low-risk/single-agent treatment, 11 (29.7%) were treated successfully, whereas 26 (70.3%) required additional multi-agent chemotherapy to achieve complete remission (CR). Combination chemotherapy was initially commenced in 28 women, and 2 (7%) required additional drugs for CR. The overall duration of therapy for those commencing and completing single- or multi-agent chemotherapy was 130 and 123 days (P=0.78), respectively. The median-treatment duration for patients commencing single-agent but changing to multi-agent chemotherapy was 13 days more than those receiving high-risk treatment alone (136 vs 123 days; P=0.07). All 3 patients with an initial hCG >400 000 IU l−1 and treated with single-agent therapy developed drug resistance. Overall survival for all patients was 100%.Conclusion:Low-risk post-molar GTN patients with a pre-treatment hCG >100 000 and <400 000 IU l−1 can be offered low-risk single-agent therapy, as this will cure 30%, is relatively non-toxic and only prolongs treatment by 2 weeks if a change to combination agents is required. Patients whose hCG is >400 000 IU l−1 should receive multi-agent chemotherapy from the outset.

Mate calling behavior of male South African clawed frogs ( Xenopus laevis) is suppressed by the antiandrogenic endocrine disrupting compound flutamide

Several environmental pollutants have been identified as antiandrogenic endocrine disrupting chemicals (EDC), with flutamide (FLU) being a model compound for this type of action. Despite impacts of EDC interfering with sexual differentiation and reproduction in amphibians, established information about suggested effects on sexual behavior is still lacking. In this study adult male Xenopus laevis were injected with human chorionic gonadotropin (hCG) to initiate mate calling behavior. After one day hCG-stimulated frogs were treated via aqueous exposure over three days without and with FLU at concentrations of 10 −8 and 10 −6 M in comparison to untreated frogs. Androgen controlled mate calling behavior was recorded during the 12 h dark period. At the end of exposure circulating levels of testosterone (T) and 17β-estradiol (E2) were determined and furthermore gene expression was measured concerning reproductive biomarkers such as hypophysial luteinizing hormone (LH), follicle-stimulating hormone (FSH), testicular aromatase (ARO), 5α reductase type 1 (SRD5α1) and 5α reductase type 2 (SRD5α2). Both concentrations of FLU caused a significant decrease in calling activity starting at the second day of exposure. HCG injected positive controls had elevated levels of T compared to negative control frogs while in parallel treatment with FLU did not affect significantly the hCG elevated sex steroid levels. Furthermore, hCG treatment led to significantly decreased levels of gene expression for ARO and SRD5α2 but no impacts were detected on LH, FSH or SRD5α1 mRNA levels compared to negative controls. In summary, the behavioral parameter mate calling is the most sensitive biomarker detecting antiandrogenic modes of action in this challenge-experiment indicating that this non-invasive method could markedly contribute for sensitive assessment of antiandrogenic EDC.

Gonadotropin‐regulated testicular RNA helicase (GRTH/DDX25) gene: Cell‐specific expression and transcriptional regulation by androgen in transgenic mouse testis

Gonadotropin-regulated testicular RNA helicase is a multifunctional enzyme present in both Leydig and germ cells that is essential for the progress of spermatogenesis. GRTH gene expression is transcriptionally upregulated by human chorionic gonadotropin (hCG) via second messenger (cAMP) and androgen in Leydig cells. The regulatory region(s) in the GRTH gene that is/are required for its cell-specific expression in the testis and hCG/androgen dependent expression were investigated in transgenic mice carrying sequential deletions of 5' flanking sequences of the GRTH gene. GFP-reporter gene expression directed by the GRTH 5' flanking sequences extending to -3.6 kb was specifically located in Leydig cells and the 205 bp minimal promoter domain was sufficient for this cell-specific expression. The 1 kb (5' to the ATG codon) transgene-directed expression was markedly increased by in vivo hCG treatment. Administration of the androgen receptor inhibitor Flutamide blocked the basal and hCG stimulated GFP expression in Leydig cells. We conclude that the expression of GRTH in testicular cells is differentially regulated by its 5' flanking sequence and that the 1 kb fragment of GRTH gene contains sequences for androgen regulation of its expression in Leydig cells.

Effects of Gonadotropins on In Vitro Maturation and of Electrical Stimulation on Parthenogenesis of Canine Oocytes

The objective of this study was to determine the effects of gonadotropins on in vitro maturation (IVM) and electrical stimulation on the parthenogenesis of canine oocytes. In experiment I, cumulus oocyte complexes were collected from ovaries at a random phase of the oestrus cycle and cultured on maturation medium treated with hCG or eCG for 48 or 72 h. There were no significant differences in the effects on the metaphase II (MII) rate between the hCG and eCG treatment groups over 48 h (5.4% vs 5.5%). The MII rate in the co-treatment group of hCG and eCG for 48 h was higher than in each hormone treated group (15.5%, p < 0.05). In experiment 2, the parthenogenetic effect on oocyte development, at various electrical field strengths (1.0, 1.5, 2.0 kV/cm DC) for 60 or 80 mus with a single DC pulse after IVM on the co-treatment of hCG and eCG, was examined. The rate of pronuclear formation (37.1%) in electrical activation at 1.5 kV/60 mus without cytochalasin B (CB) was higher than that of oocytes activated in the other groups (p < 0.05). However, we did not observe the cleavage stages. Also, CB did not influence parthenogenesis of canine oocytes. The results showed that the pronucleus formation rate, indicative of the parthenogenesis start point, could be increased by electrical stimulation. Therefore, these results can provide important data for the parthenogenesis of canine oocytes and suggest the probability of parthenogenesis in canines.

Attenuation of cell cycle progression by 2,3,7,8-tetrachlorodibenzo-p-dioxin eliciting ovulatory blockade in gonadotropin-primed immature rats

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) reduces ovulation rate in rats. The present study was to investigate whether TCDD alters the progression of cell cycle, and thus resulting in the blockade of ovulation in gonadotropin-primed, immature rats. The ovulation rate and ovarian weight were reduced in intact rats given TCDD (32 µg/kg BW in corn oil) by gavage one day before pregnant mare's serum gonadotropin (PMSG; 5 IU/rat) injection. Flow cytometry demonstrated that the percentage of granulosa cells in S-phase was increased at 24 h following PMSG treatment, but declined at 8 h following hCG treatment in corn oil-treated rats. Interestingly, the number of S-phase cells in TCDD-treated rats was reduced 24 and 48 h following PMSG treatment. TCDD, however, increased the percentage of cells in G2/M-phase at 24 h following PMSG treatment. TCDD inhibited the mRNA levels of Cdk2 at 0 h and 24 h, and cyclin D2 at 24 h and 48 h following PMSG treatment. Protein levels of aryl hydrocarbon receptor in granulosa cells were elevated in TCDD-treated rats at 12 h and 24 h following PMSG treatment. The present study indicates that TCDD reduces S-phase cells and inhibits levels of Cdk2 and cyclin D2 at 24 h following PMSG treatment, implying the ovulation-inhibiting action of TCDD may be exerted through the attenuation of cell cycle progression via AhR-mediated cascade.

Investigation of preovulatory serum progesterone increase after hCG treatment in mares

Investigation of preovulatory serum progesterone increase after hCG treatment in mares

The involvement of hypophyseal‐gonadal and hypophyseal‐adrenal axes in arsenic‐mediated ovarian and uterine toxicity: Modulation by hCG

This study evaluated the involvement of hypophyseal-gonadal and hypophyseal-adrenal axes as a possible mechanism of sodium arsenite toxicity in ovary and uterus by the coadministration of hCG. Subchronic treatment of 0.4 ppm of sodium arsenite/(100 g body weight day) via drinking water for seven estrous cycles significantly suppressed the plasma levels of leutinizing hormone, follicle-stimulating hormone, and estradiol along with sluggish ovarian activities of Delta(5),3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase followed by a reduction in gonadal tissue peroxidase activities in mature female rats at diestrous phase. Noticeable weight loss of the ovary and uterus along with prolonged diestrous phase and increased deposition of arsenic in the plasma and in these reproductive organs were also demonstrated following the ingestion of arsenic. Follicular atresia and thinning of the uterine luminal diameter were evident after sodium arsenite treatment. Effective protection of gonadal weight loss, suppressed ovarian steroidogenesis, and altered ovarian and uterine peroxidase activities were noticed when 1.0 IU hCG/(100 g body weight day) is given in arsenic-intoxicated rats. Normal estrous cyclicity was restored toward the control level after hCG coadministration, though the elimination of elementary arsenic from the plasma and gonadal tissues was impossible. A significant recovery in the restoration of ovarian and uterine histoarchitecture was prominent after hCG treatment. Adrenal hypertrophy and steroidogenic arrest of the adrenal gland along with altered level of brain monoamines in the midbrain and diencephalons following arsenic intoxication were also ameliorated after hCG coadministration.

422 GENERATION OF REPORTER TRANSGENIC MICE FOR THE CHEMOKINE CXCL2 USING TWO DIFFERENT DNA CONCENTRATIONS

Transgenic mice have important implications in biomedicine, and are widely employed to understand gene functions and their regulation. The improvement of transgenic efficiency is relevant because of the low rate of success for this technology. CXCL2 is a chemokine secreted by macrophages and epithelial cells under proinflammatory stimulus of the innate immune response such as bacterial endotoxins. The main effect of CXCL2 is the recruitment of neutrophils to the site of production to fight infections. The objective of this study was to evaluate the effect of 2 DNA concentrations in the efficiency of the transgenesis process. To this aim we used a luciferase reporter under the control of CXCL2 promoter for the generation of a transgenic line to report activation of innate immune response. A total of 1727 1-cell embryos were divided into 2 experimental groups to be microinjected with 0.5 or 1.0 ng μL-1 of DNA in 25 sessions. Three-week-old B6SJL F1 females (n = 131) were superovulated with 5 IU of eCG i.p. (Novormon, Syntex, Buenos Aires, Argentina) and 5 IU of hCG i.p. (Ovusyn, Syntex) 46 h later, and mated with B6SJL F1 stud males. At the moment of hCG treatment, foster females were mated with vasectomized males to induce pseudogestation. Donor females were euthanized by cervical dislocation 20 h after hCG treatment, and embryos were recovered from the ampulla, denuded in 300 μg mL-1 hyaluronidase (Sigma, St. Louis, MO, USA) and incubated at 37°C with 5% CO2, in drops of M16 media (Sigma) under mineral oil, until microinjection. DNA construction consisted of the luciferase reporter gene under the control of the murine CXCL2 gene promoter. Embryos were microinjected into 1 pronucleus under an inverted microscope (Nikon, NY, USA) using glass microtools and mechanic micromanipulators (Eppendorf, Hamburg, Germany). Intact/injected embryos were assessed 30 min after microinjection. Fifteen to 20 embryos per foster female were transferred in both oviducts. Birth rate, survival of pups at Day 7 after birth, number of transgenic pups assessed by standard PCR, and overall transgenic efficiency was registered for each group. Data were analyzed by Yates-corrected chi-square test. No statistical differences were founded except for a higher number of pups alive/embryo transferred in the lower DNA concentration, suggesting the advantage of using 0.5 ng μL-1 v. 1.0 ng μL-1. Table 1.Effect of DNA concentration in the generation of CXCL2-luc transgenic mice

Avaliação de protocolos para indução de inatividade ovariana em gatas domésticas

Os índices de prenhez após inseminação artificial em felídeos selvagens não são satisfatórios devido ao variável ambiente endócrino após a estimulação com gonadotropinas. O objetivo deste estudo consistiu em aumentar a taxa de sucesso em programas de inseminação artificial em gatas domésticas (animal modelo). As fêmeas (n=9) foram divididas em três grupos, cada um com três animais, sendo: 1) controle (C), somente 200 UI eCG/ 100 UI hCG ; 2) levonorgestrel oral (L) (0,075 mg) durante 37 dias + eCG/hCG; 3) etonogestrel (E), implante subdérmico durante 37 dias + eCG/hCG. Foram submetidas ao exame laparoscópico 29-39 horas após a administração de hCG para verificação da resposta ovariana e realização de esfregaço vaginal para monitoração da fase do ciclo estral. Foram coletadas amostras de fezes 60 dias antes e 60 dias após o tratamento com gonadotropinas para dosagem hormonal de estrógenos. Os resultados foram avaliados através do Teste ANOVA. Os níveis de significância mostraram que o Grupo E, em contraste com o Grupo C e o Grupo L, apresentou inibição satisfatória das concentrações de estrógenos durante a sua utilização. O grupo L não apresentou inibição ovariana durante o tratamento e diferença significativa em relação ao Grupo C. No exame laparoscópico todas as fêmeas dos grupos C, L e E apresentaram folículos e 77% das fêmeas apresentaram corpo lúteo. Também apresentaram células epiteliais superficiais anucleadas e nucleadas características de estro. Concluiu-se que a utilização de implantes de etonogestrel em gatas domésticas mostrou-se eficaz, possibilitando a sua utilização prévia aos programas de inseminação artificial, aspiração folicular e também para a contracepção.

Human chorionic gonadotropin (hCG) prevents the transformed phenotypes induced by 17 beta-estradiol in human breast epithelial cells.

Human chorionic gonadotropin (hCG), a hormone produced during pregnancy, can elicit life-long refractoriness to carcinogenesis by differentiation of the breast epithelium. Human breast epithelial cells MCF-10F form tubules in collagen, mimicking the normal ductules. We have shown that 17 beta-estradiol (E2) alter the ductulogenic pattern of these cells. The effect of the recombinant hCG (rhCG) in vitro was evaluated on the transformation of MCF-10F induced by E2. MCF-10F cells were treated with 70 nM E2 alone or in combination with 50 IU/ml rhCG during 2 weeks, while the controls were treated with DMSO (the solvent in which E2 was dissolved) or rhCG alone. At the end of treatment, the cells were plated in type I collagen matrix (3D-cultures) for detecting 2 main phenotypes of cell transformation, namely the loss of ductulogenic capacity and the formation of solid masses. Although E2 significantly increased solid mass formation, this effect was prevented when MCF-10F cells were treated with E2 in combination with rhCG. Furthermore, E2 increased the main duct width (p < 0.001), and caused a disruption of the luminal architecture, whereas rhCG increased the length of the tubules (p < 0.001) and produced tertiary branching. In conclusion, rhCG was able to abrogate the transforming abilities of estradiol, and had the differentiating property by increasing the branching of the tubules formed by breast epithelial cells in collagen. These results further support our hypothesis, known as the terminal differentiation hypothesis of breast cancer prevention, that predicts that hCG treatment results in protection from tumorigenic changes by the loss of susceptible stem cells 1 through a differentiation to refractory stem cells 2 and increase differentiation of the mammary gland.

Production of Boer goat (Capra hircus) by nuclear transfer of cultured and cryopreserved fibroblast cells into slaughterhouse-derived oocytes

The present study reports a birth of goat clone produced by nuclear transfer of cultured and cryopreserved fibroblast cells into slaughterhouse-derived oocytes. The donors of somatic cells were fibroblasts derived from the ear skin of a Boer goat while the recipient ooplasm was &lt;I&gt;in vitro&lt;/I&gt; matured oocytes of Huanghuai white goat, an Anhui native goat species. The reconstructed embryos were cultured &lt;I&gt;in vitro&lt;/I&gt; and then the morphologically normal embryos were transferred to the surrogates. The reconstructed embryos were surgically transferred into 37 recipient surrogates, Huanghuai white goats with natural oestrus. Five of them were treated with hCG after transfer. Among them, one was pregnant and gave birth to a live kid. Due to the improper delivery aid, the cloned kid died accidentally after birth. The cloned kid was then anatomised to observe the viscera development, and the results showed that the organs were normal. Paraffin tissue slices were prepared and stained to ensure the viscera development further, and the results suggested that the organs also developed well in spite of incipient hydropericardium. The microsatellite analysis identified the cloning. It is suggested that the optimised nuclear transfer protocol and proper hCG treatment lead to the successful birth of a goat clone.

Regulation of milk fat globule epithelial growth factor eight (MFG-E8) by human chorionic gonadotropin (HCG) in the human endometrium

OBJECTIVE: MFG-E8, a protein involved in breast tissue remodeling, has recently been demonstrated to be present in the human endometrium. One of the earliest hormones secreted by the embryo is hCG, proposed to be involved in tissue changes integral to implantation. The objective of this study was to test the hypothesis that hCG regulates MFG-E8 mRNA expression in the human endometrium. DESIGN: Experimental MATERIALS AND METHODS: Epithelial and stromal cells from peri-ovulatory endometrial biopsies (n=3) were isolated and primary cultures established. The presence of the hCG receptor protein and mRNA was confirmed by immunocytochemistry and PCR. Cells were cultured to 80% confluence and treated with escalating doses of highly purified hCG for 6 hours and recombinant MFG-E8 for 24 hours. MFG-E8 mRNA gene expression was analyzed using real-time RT PCR. Caspase 3 and 7 activity after MFG-E8 treatment was measured with a luciferase activation assay. Means were compared with the Mann-Whitney test. RESULTS: hCG resulted in a statistically significant (P<.05) decreased gene expression of MFG-E8 in both the stromal and epithelial compartments at all treatment doses. In the stromal cells, MFG-E8 was significantly down regulated after hCG treatment by 22% (0.5 mIU/mL), 28% (5 mIU/mL), 36% (50 mIU/mL), and 24% (500 mIU/mL). In the epithelial cells, MFG-E8 was significantly down regulated after hCG treatment by 12% (5 mIU/mL), 20% (50 mIU/mL), and 26% (500 mIU/mL). MFG-E8 treatment of stromal cells resulted in a 13-fold increase in caspase activity (P=0.037). CONCLUSIONS: In cultured peri-ovulatory human endometrial cells, hCG induces a decrease in MFG-E8 gene expression. MFG-E8 may have pro-apoptotic activity in the human endometrium. We propose that hCG secreted by the early embryo may play a role in tissue remodeling and apoptosis to facilitate implantation.