Pregnant Mare Serum Gonadotropin Injection Research Articles

Novel In Vivo Evidence That Not Only Androgens But Also Pituitary Gonadotropins and Prolactin Directly Stimulate Murine Bone Marrow Stem Cells – Implications For Potential Treatment Strategies In Aplastic Anemias

BackgroundHematopoietic stem/progenitor cells (HSPCs) are exposed in vivo to several growth factors, cytokines, chemokines, and bioactive lipids in bone marrow (BM) in addition to various sex hormones circulating in peripheral blood (PB). It is known that androgen hormones (e.g., danazol) is employed in the clinic to treat aplastic anemia patients. However, the exact mechanism of action of sex hormones secreted by the pituitary gland or gonads is not well understood. Therefore, we performed a complex series of experiments to address the influence of pregnant mare serum gonadotropin (PMSG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), androgen (danazol) and prolactin (PRL) on murine hematopoiesis. In particular, from a mechanistic view we were interested in whether this effect depends on stimulation of BM-residing stem cells or is mediated through the BM microenvironment. Materials and MethodsTo address this issue, normal 2-month-old C57Bl6 mice were exposed or not to daily injections of PMSG (10 IU/mice/10 days), LH (5 IU/mice/10 days), FSH (5 IU/mice/10 days), danazol (4 mg/kg/10 days) and PRL (1 mg/day/5days). Subsequently, we evaluated changes in the BM number of Sca-1+Lin–CD45– that are precursors of long term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2011;25:1278–1285) and bone forming mesenchymal stem cells (Stem Cell & Dev. 2013;22:622-30) and Sca-1+Lin–CD45+ hematopoietic stem/progenitor cells (HSPC) cells by FACS, the number of clonogenic progenitors from all hematopoietic lineages, and changes in peripheral blood (PB) counts. In some of the experiments, mice were exposed to bromodeoxyuridine (BrdU) to evaluate whether sex hormones affect stem cell cycling. By employing RT-PCR, we also evaluated the expression of cell-surface and intracellular receptors for hormones in purified populations of murine BM stem cells. In parallel, we studied whether stimulation by sex hormones activates major signaling pathways (MAPKp42/44 and AKT) in HSPCs and evaluated the effect of sex hormones on the clonogenic potential of murine CFU-Mix, BFU-E, CFU-GM, and CFU-Meg in vitro. We also sublethally irradiated mice and studied whether administration of sex hormones accelerates recovery of peripheral blood parameters. Finally, we determined the influence of sex hormones on the motility of stem cells in direct chemotaxis assays as well as in direct in vivo stem cell mobilization studies. ResultsWe found that 10-day administration of each of the sex hormones evaluated in this study directly stimulated expansion of HSPCs in BM, as measured by an increase in the number of these cells in BM (∼2–3x), and enhanced BrdU incorporation (the percentage of quiescent BrdU+Sca-1+Lin–CD45– cells increased from ∼2% to ∼15–35% and the percentage of BrdU+Sca-1+Lin–CD45+ cells increased from 24% to 43–58%, Figure 1). These increases paralleled an increase in the number of clonogenic progenitors in BM (∼2–3x). We also observed that murine Sca-1+Lin–CD45– and Sca-1+Lin–CD45+ cells express sex hormone receptors and respond by phosphorylation of MAPKp42/44 and AKT in response to exposure to PSMG, LH, FSH, danazol and PRL. We also observed that administration of sex hormones accelerated the recovery of PB cell counts in sublethally irradiated mice and slightly mobilized HSPCs into PB. Finally, in direct in vitro clonogenic experiments on purified murine SKL cells, we observed a stimulatory effect of sex hormones on clonogenic potential in the order: CFU-Mix > BFU-E > CFU-Meg > CFU-GM. ConclusionsOur data indicate for the first time that not only danazol but also several pituitary-secreted sex hormones directly stimulate the expansion of stem cells in BM. This effect seems to be direct, as precursors of LT-HSCs and HSPCs express all the receptors for these hormones and respond to stimulation by phosphorylation of intracellular pathways involved in cell proliferation. These hormones also directly stimulated in vitro proliferation of purified HSPCs. In conclusion, our studies support the possibility that not only danazol but also several other upstream pituitary sex hormones could be employed to treat aplastic disorders and irradiation syndromes. Further dose- and time-optimizing mouse studies and studies with human cells are in progress in our laboratories.▪ Disclosures:No relevant conflicts of interest to declare.

12 PHARMACOLOGICAL STIMULATION OF OVULATION IN THE BLACK FLYING-FOX (PTEROPUS ALECTO)

Artificial insemination (AI) involves the accurate detection of oestrus, the ability to pharmacologically induce ovulation, or both. This is particularly important in flying-foxes (Pteropus spp.) that do not have an overt behavioural oestrus. Pregnant mare serum gonadotropin (PMSG) stimulates folliculogenesis and ovulation in the Little Red Flying-fox (P. scapulatus; Towers PA and Martin L 1985 Proc. Aust. Soc. Reprod. Biol. 17, 115). In this study, a dose rate of 15 IU PMSG was used. Our study investigated if 15 IU PMSG would sufficiently induce ovulation in the larger, Black Flying-fox (P. alecto) or if a higher dose (30 IU) would be necessary. Before the mating season, a single injection of PMSG (Folligon®, Intervet Australia Pty Ltd, Bendigo East, Australia) was administered i.m. to eight adult females at 15 IU (n = 4) or 30 IU (n = 4) on Day 0. On Day 4, semen was collected by electro-ejaculation from adult males and inseminated intravaginally into females under isoflurane (Forthane®, Abbott Australasia Pty Ltd, Sydney, Australia) anaesthesia. Ovaries and reproductive tracts were surgically removed on Day 6, fixed, serially sectioned and stained using Gomori’s Stain. Histological sections were examined for evidence of ovarian activity and the presence of ova and spermatozoa in the reproductive tracts. Preliminary observations showed evidence of ovulation in both groups in the form of at least one CL in either ovary and an ovum in the ipsilateral oviduct or uterine horn. Ovaries of females stimulated with 30 IU PMSG differed noticeably from those treated with 15 IU in containing multiple, large, collapsed luteinized follicles with retained oocytes. Vascularization and glandular hypertrophy of the endometrium was also more evident in the higher dose group. No spermatozoa were observed in any of the excised tracts. These results suggest that whereas both doses of PMSG induce ovulation, the administration of 30 IU PMSG may over-stimulate the ovaries. This, in turn, could lead to an unphysiological environment for successful fertilization and embryonic development. A dose level of 15 IU or slightly above may be sufficient for subsequent attempts to stimulate folliculogenesis and ovulation in P. alecto. The absence of spermatozoa suggests that the site of insemination, the number of spermatozoa inseminated, or both requires further investigation if AI is to be successfully implemented in these species. We conclude that the ovarian responses to PMSG indicate that pharmacological induction of ovulation can be successfully achieved and thereby utilized in AI programs of endangered Pteropus species.

Complete follicular development and recovery of ovarian function of frozen-thawed, autotransplanted caprine ovarian cortex

Frozen-thawed ovarian cortical fragments (1 mm(3)) were autotransplanted to the uterus of completely ovariectomized goats. The grafts developed preovulatory follicles, accompanied by estrous behavior and a rise in plasma E(2) levels, demonstrating successful cryopreservation and transplantation.

Concentrations de l’hormone lutéinisante et de la progestérone chez des vaches zébus Azawak soumises à différents protocoles de synchronisation de l’oestrus

Quinze vaches zébus Azawak ont été soumises à deux protocoles de synchronisation de l’oestrus. Les concentrations plasmatiques de LH et le pourcentage des femelles ayant exhibé un comportement d’oestrus ont été répertoriés. Le premier traitement de synchronisation (T1, n = 9) a été effectué avec des implants sous-cutanés de norgestomet placés sous la peau de l’oreille pendant 10 jours. Le traitement a été complété par des injections de valérate d’oestradiol, de prostaglandine F2α et de pregnant mare serum gonadotropin (PMSG). Le deuxième protocole (T2, n = 6) a consisté en deux injections de prostaglandine F2α à 11 jours d’intervalle, suivies d’une injection de PMSG deux jours après la deuxième injection de prostaglandine. Des échantillons de sang ont été prélevés à trois heures d’intervalle pendant cinq jours après le retrait de l’implant (T1) ou l’injection de PMSG (T2) pour mesurer les taux de LH. Le taux d’oestrus induit a été plus élevé chez les vaches du groupe T2, mais cette différence n’a pas été significative. En général, le temps observé entre la fin du traitement et le début de l’oestrus a été plus court chez les femelles traitées au norgestomet (35,9 ± 3,9 h) que chez celles traitées à la prostaglandine (49,5 ± 5,8 h). Sept sur les neuf femelles traitées au norgestomet et seulement deux sur les six traitées à la prostaglandine ont présenté un pic de LH. L’intervalle moyen entre la fin des traitements et le pic de LH a eu tendance à être plus long chez les femelles traitées à la prostaglandine que chez celles traitées au norgestomet. Une femelle a présenté des concentrations de LH anormalement élevées après le retrait de l’implant. Toutefois, aucun pic de LH n’a été observé pendant la période de suivi.

Human menopausal and pregnant mare serum gonadotrophins in murine superovulation regimens for transgenic applications

Superovulation is a fundamental procedure for generating transgenic rodents. While various methods exist, zygote yield/quality remain suboptimal, making these techniques open to refinement. All require a follicle stimulating and a luteinising effect. The former can be induced by pregnant mare serum gonadotrophin (PMSG) or other compounds like human menopausal gonadotrophin (HMG). While HMG can double zygote yield compared to PMSG, no study has compared their effects on embryo quality. Embryo yield could also be increased with PMSG: timing administration at estrus may further improve follicular recruitment. This study compared: (i) the efficacy of HMG/PMSG for producing viable embryos for microinjection; and (ii) the effect of HMG/PMSG administration at estrus on embryo yield. Whitten effect-induced estrous C57/Bl6xCBA F 1 hybrid mice were superovulated as follows: PMSG (day 1; 5 IU intraperitoneally) or HMG (days 1 and 2; 1 IU intramuscularly); all received human chorionic gonadotrophin (hCG) on day 3 (5 IU, intraperitoneally). Zygotes were retrieved following mating, morphologically assessed and microinjected with innocuous ZhAT1R construct (expressing LacZ reporter and human angiotensin II type 1 receptor) before transfer to pseudopregnant recipients. Pups were tested for the transgene by Southern blot. Neither HMG nor PMSG proved superior in improving embryo yield, morphology and short-term post-microinjection survival. However, HMG group micromanipulated embryos all failed to establish a pregnancy/generate transgenic pups, unlike their PMSG counterparts. While HMG can be used for superovulation, it appears to increase embryo vulnerability to the long-term effects of microinjection. Furthermore, the embryo yields associated with HMG can be replicated by timing PMSG injection to coincide with Whitten effect-induced estrus.

Polyploidy in mouse embryos derived from in vivo and in vitro fertilization is dependent on the timing of pregnant mare serum gonadotropin (PMSG) injection

Nine hundred eighty-seven mouse embryos were cytogenetically analyzed at the two-cell stage to compare aneuploidy rates in embryos derived from both in vivo and in vitro fertilization using two different ovarian stimulation protocols. The rate of polyploidy was significantly (P<.001) elevated in both in vivo fertilized (15.6%) and in vitro fertilized (5.9%) embryos when PMSG injection was given in the afternoon instead of the morning (2.1% and 4.0% respectively).

Gonadotropins affect Oct‐4 gene expression during mouse oocyte growth

The transcription factor Oct-4 is required for the maintenance of stem cells pluripotency and is involved in the regulation of the expression of a number of developmental genes. Oct-4 is also expressed in the female gamete during folliculogenesis, but the role it plays is largely unknown. Its upstream and promoter regions have some characteristic features that make this gene a possible target of hormonal regulation. To further our understanding of Oct-4 gene expression during oocyte growth, we tested whether changes to the hormonal milieu of the ovary may affect its transcription. Using a semi-quantitative single-cell-sensitive reverse transcription-polymerase chain reaction (RT-PCR) assay, we investigated the pattern of Oct-4 expression during mouse oocyte growth in females intraperitoneally injected with pregnant mare serum gonadotropin (PMSG) alone or PMSG followed by human chorionic gonadotropin (hCG). The results of this study show that gonadotropins induced two major increases in Oct-4 expression during folliculogenesis: (1) 48 hr after PMSG injection, in oocytes isolated from primordial follicles; and (2) following a surge of hCG, in preovulatory antral oocytes. These results suggest a potential twofold role for this gene in the recruitment of oocytes for initiating growth and in the selection of oocytes for ovulation. Also, they may contribute to our knowledge of the molecular bases of oocyte growth, meiosis resumption, and acquisition of a developmental competence.

Gonadotropin-Induced Superovulation Drives Ovarian Surface Epithelia Proliferation in CD1 Mice

The ovarian surface epithelium (OSE) is a monolayer of cells that surround the ovary and accommodate repeated tear and repair in response to ovulation. OSE cells are thought to be the progenitors of 90% of ovarian cancers. Currently, the total amount of proliferation of the OSE has not been reported in response to one ovulatory event. In this study, proliferation of the OSE was quantified in response to superovulation induced by ip injection of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) in immature 27-d-old CD1 mice using bromodeoxyuridine (BrdU). BrdU incorporation into the OSE cells was measured from the time of hCG injection for a total cumulative label of 12 h. BrdU incorporation was also measured from the time of PMSG injection for a total label of 60 h to correlate proliferation with specific gonadotropin stimulation. The OSE proliferation was significantly higher in superovulated animals compared with control mice at all time points. Proliferation was also analyzed in discrete anatomical sections and indicated that OSE covering antral follicles and corpora lutea proliferated more rapidly than OSE distal to follicular growth. Finally, apoptosis was assessed in response to ovulation, and virtually no cell death within the OSE was detected. These data demonstrate that the OSE, especially near antral follicles and corpora lutea, proliferates significantly in response to the gonadotropins PMSG and hCG. Therefore, ovarian surface cell division in response to ovulation could contribute to ovarian cancer by proliferation-induced DNA mutations and transformed cell progression.

Gonadotropin regulation of glutamate cysteine ligase catalytic and modifier subunit expression in rat ovary is subunit and follicle stage specific

We have observed that levels of the antioxidant glutathione (GSH) and protein levels of the catalytic and modifier subunits of the rate-limiting enzyme in GSH synthesis, GCLc and GCLm, increase in immature rat ovaries after treatment with gonadotropin. The goals of the present studies were to delineate the time course and intraovarian localization of changes in GSH and GCL after pregnant mare's serum gonadotropin (PMSG) and after an ovulatory gonadotropin stimulus. Twenty-four hours after PMSG, there was a shift from predominantly granulosa cell expression of gclm mRNA, and to a lesser extent gclc, to predominantly theca cell expression. GCLc immunostaining increased in granulosa and theca cells and in interstitial cells. Next, prepubertal female rats were primed with PMSG, followed 48 h later by 10 IU of hCG. GCLm protein and mRNA levels increased dramatically from 0 to 4 h after hCG and then declined rapidly. There was minimal change in GCLc. The increase in gclm mRNA expression was localized mainly to granulosa and theca cells of preovulatory follicles. To verify that GCL responds similarly to an endogenous preovulatory gonadotropin surge, we quantified ovarian GCL mRNA levels during the periovulatory period in adult rats. gclm mRNA levels increased after the gonadotropin surge on proestrus and then declined rapidly. Finally, we assessed the effects of gonadotropin on ovarian GCL enzymatic activity. GCL enzymatic activity increased significantly at 48 h after PMSG injection and did not increase further after hCG. These results demonstrate that gonadotropins regulate follicular GCL expression in a follicle stage-dependent manner and in a GCL subunit-dependent manner.

Heat stress diminishes gonadotropin receptor expression and enhances susceptibility to apoptosis of rat granulosa cells

Heat stress inhibits ovarian follicular development in mammalian species. We hypothesized that heat stress inhibits the function of follicular granulosa cells and suppresses follicular development. To test this, immature female rats were injected with pregnant mare serum gonadotropin (PMSG) at 48 h after the start of temperature treatment (control: 25 degrees C, 50% RH; heat stress: 35 degrees C, 70% Relative Humidity). The ovaries and granulosa cells of follicles at different developmental stages were analyzed for gonadotropin receptor levels and aromatase activity; estradiol levels were measured in follicular fluid. Before injection, heat stress diminished only the amount of FSH receptor on granulosa cells of antral follicles. During PMSG-stimulated follicular development, heat stress strongly inhibited gonadotropin receptor levels and aromatase activity in granulosa cells, and estradiol levels in the follicular fluid of early antral, antral and preovulatory follicles. To examine apoptosis and mRNA levels of bcl-2 and bax in granulosa cells, follicles harvested 48 h after PMSG injection were cultured in serum-free conditions. Heat-stressed granulosa cells showed a time-dependent increase in apoptosis. The bcl-2 mRNA levels were similar in control and heat-stressed granulosa cells; bax mRNA levels were increased in heat-stressed granulosa cells. According to these results, heat stress inhibits expression of gonadotropin receptors in granulosa cells and attenuates estrogenic activity of growing follicles, granulosa cells of heat-stressed follicles are susceptible to apoptosis, and the bcl2/bax system is not associated with heat-stress-induced apoptosis of granulosa cells. Our study suggests that decreased numbers and function of granulosa cells may cause ovarian dysfunction in domestic animals in summer.

EFISIENSI PRODUKSI SUSU SAPI PERAH DARA DAN LAKTASI AKIBAT PENYUNTIKAN PMSG

The aim of the presentexperiment was to evaluate the influence of I 500 TU injection of Pregnant Mare Serum Gonadotrophin (PMSG) at the mating program in the Fresian Holstein (FH) dairy cow on the performancesof milk production, quality, and production efficiency. The experimentwas performed at Banaran Village, Cepogo Subdistrict ofBoyolali District used twelve heifers and the-pregnant first parity cow of about 350-450 kg body weight. A split-plot experimental design with heifer (Pol and first parity (P1) as a main plot, and PMSG doses (T0 and T ,)as a sub plot. Thus the treatmentwere I) heifer with no PSMG injection (P0T0), 2) PMSG injected heifer (P0T,), 3) first parity cow with no PMSG injection, and4) PMSG injected first parity cow P,T,). Measurements were conducted on 90 days milk production, mass weight of milk, fat and lactose contents, and milk production efficiency. Results showed that average milk productions of not injected (804.67 l) and injected PMSG heifers ( 1,286.92 l) were significantly different (P 0.05) influenced average milk productions of first parity cows ( J.0491 versus 1,4832.52 J, for not injected and injected PMSG cow, respectively). Fat production of the not injected and injected PMSG heifers were 25.27 and 41.12 kg (P 0.05). Milk production efficiency of not injected and injected PMSG heifers were 11.13 and 18.45%, respectively (P 0.05). In general there were no significant differenton theeffectof PMSG injection on milk quality components (mass weight, fat and lactose contents). In conclusion, in the present experiment PMSG injection improved milk pro• duction and it efficiency, but did not improved milk quality.

Changes in mouse granulosa cell gene expression during early luteinization.

Changes in gene expression during granulosa cell luteinization have been measured using serial analysis of gene expression (SAGE). Immature normal mice were treated with pregnant mare serum gonadotropin (PMSG) or PMSG followed, 48 h later, by human chorionic gonadotropin (hCG). Granulosa cells were collected from preovulatory follicles after PMSG injection or PMSG/hCG injection and SAGE libraries generated from the isolated mRNA. The combined libraries contained 105,224 tags representing 40,248 unique transcripts. Overall, 715 transcripts showed a significant difference in abundance between the two libraries of which 216 were significantly down-regulated by hCG and 499 were significantly up-regulated. Among transcripts differentially regulated, there were clear and expected changes in genes involved in steroidogenesis as well as clusters of genes involved in modeling of the extracellular matrix, regulation of the cytoskeleton and intra and intercellular signaling. The SAGE libraries described here provide a base for functional investigation of the regulation of granulosa cell luteinization.

Low-dose LH administration induces ovulation and prevents vascular hyperpermeability and VEGF expression in superovulated rats

Objective: The combined use of Pregnant Mare Serum Gonadotropin (PMSG) and hGC in high doses induces ovarian hyperstimulation syndrome (OHSS) in the rat animal model, characterized by increased vascular permeability related to the simultaneous overexpression of vascular endothelial growth factor (VEGF) and its receptor 2 in ovarian cells. hCG has longer half-life than LH and is believed to have a higher biological activity (approximately 6-fold), including the incidence of complications such as OHSS. Similarly, FSH may also be related to the ovulatory changes within the follicle because there is a simultaneous surge in spontaneous cycles. The aim of this study was to compare the ability of hCG, FSH, and LH in inducing ovulation, and simultaneously prevent OHSS in the animal model. Design: Immature female rats, weighing 42–48 g at the beginning of the experiments were treated daily with 10 IU PMSG injections for 4 consecutive days, and the fifth day 10 IU hCG (hCG group), 10 IU FSH (FSH group), 10 IU LH (LH-10 group), 60 IU LH (LH-60), or saline (control group), to induce ovulation. A total of 30 animals per group were employed in three sets of experiments. In each set and group, half of the animals were sacrificed 12 hours after ovulation triggering to ascertain the number of ovulated oocytes. The remaining rats were employed to measure vascular permeability (VP) 48 hrs after administration of the drugs inducing ovulation. The ovaries were removed and frozen at −80 °C for VEGF mRNA analysis. Materials and Methods: The cumulus complexes obtained 12 hours after ovulation were disolved with hyaluronidase (1%) to ascertain the number of oocytes. To measure VP, 0.2 ml 5mM Evans blue(EB) were injected via the femoral vein. After 30 min, the peritoneal cavity was irrigated with saline. Light absorption was measured in the recovered fluid at 595 nm and the VP expressed as μg EB/100 g body weight. To analyze VEGF expression, RNA was extracted from 4 ovaries in each group and reverse transcribed. B-actin and VEGF primers were used under universal conditions to perform a QF-PCR with an ABIPRISM 7700 thermocycler. A six-point calibration curve was run in parallel to quantify VEGF expression as the VEGF/B-actin ratio. Results: hCG (27.2±2.5), LH-60 (26.7±4.1), FSH (30.4±3.2), and LH-10 (20.0±1.8) had significantly (p < 0.05) higher number of oocytes ovulated than the control group (0.7±0.1). The level of extravasated EB was 30.7±4.1μg EB/100 g body weight in the hCG group, similar to that of FSH (28.2±3.0) and LH-60 (21.1±3.7), and significantly higher than in the LH-10 (12.5±2.3, p = 0.006) and the control (10.8±1.9, p = 0.03) groups. VEGF expression was associated with increased VP, showing similar values in the hCG (1.9±0.2), LH-60 (1.4±0.3), and FSH (1.7±0.3) groups, whereas it was significantly reduced in the LH-10 (1.2±0.2, p = 0.02) and control (1.1±0.3, p = 0.03) groups. Conclusions: FSH and hCG, as well as a 6-fold increase in LH administration, have similar biological activities, including increased VP due to excessive VEGF expression. The use of lower LH doses reaches similar rates of ovulation while preventing the undesired changes in permeability. Thus, these experiments should encourage clinicians to find the optimal dose of LH to be employed in women to trigger ovulation and simultaneously avoid the risk of OHSS.

Effect of Superovulation on the Expression of Tissue Inhibitor of Metalloproteinase-3 in the Murine Endometrium

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important in the endometrial remodeling process during embryonic trophoblast invasion. Previous studies, using in situ hybridization, revealed that the predominant proteinase expressed by the implanting mouse embryo is matrix metalloproteinase-9 (MMP-9), and the major tissue inhibitor of metalloproteinases (TIMP) produced in the murine endometrium during this period is TIMP-3. Quantitative, competitive-reverse transcription-PCR was performed to evaluate the expression of TIMP-3 mRNA during the implantation period in the hyperstimulated murine endometrium from pregnant uteri of gestation day (g.d.) 6.0 and 7.0 after injection of 5 and 10 IU pregnant mare serum gonadotropin (PMSG). In PMSG groups, the expression of TIMP-3 mRNA, normalized by MMP-9 mRNA, showed a significant increase compared to the control group (natural coitus without PMSG injection) on g.d. 6.0 (p = 0.039) and an increasing tendency on g.d. 7.0. This study suggests that ovarian hyperstimulation by gonadotropin may have a detrimental effect on embryonic implantation possibly via an altered endometrial remodeling process by the increase in expression of TIMP-3 in the murine endometrium.

Effect of superovulation on the expression of tissue inhibitor of metalloproteinases-3 (TIMP-3) in murine endometrium

Objective: Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important in endometrial remodeling process during embryonic trophoblast invasion. Previous studies, using in situ hybridization, revealed that the predominant proteinase expressed by the implanting mouse embryo is matrix metalloproteinase-9 (MMP-9), and the major tissue inhibitor of metalloproteinases (TIMP) produced in murine endometrium during this period is TIMP-3. This study was performed to evaluate the expression of TIMP-3 mRNA during periimplantation period in the hyperstimulated murine endometrum.Design: Laboratory research.Materials/Methods: Quantitative, competitive-reverse transcription-polymerase chain reaction (QC-RT-PCR) was performed to evaluate the expression of TIMP-3 mRNA during periimplantation period in the hyperstimulated murine endometrum from pregnant uteri of gestation day (g.d.) 6.0 and 7.0 after injection of pregnant mare serum gonadotropin (PMSG) 5 IU and 10 IU. TIMP-3 mRNA expression was corrected by the simultaneous transcript level of matrix metalloproteinases-9 (MMP-9) which is mainly expressed by mouse embryos, since TIMP-3 is expressed mainly in the murine endometrium adjacent to implanting mouse embryos.Results: In both PMSG 5 IU and 10 IU groups, the expression of TIMP-3 mRNA showed an increase while MMP did not show a significant difference compared to control group (natural coitus without PMSG injection), on g.d. 6.0 and 7.0. Corrected TIMP-3 mRNA expression for MMP-9 also showed a high level of transcription in murine endometrium in the superovulation groups.Conclusions: This study suggests that ovarian hyperstimulation by gonadotropin, which produces many oocytes and embryos, may have a detrimental effect on embryonic implantation via its relevant endometrial remodeling process by the increase in expression of TIMP-3 in murine endometrium.Supported by: None. Objective: Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important in endometrial remodeling process during embryonic trophoblast invasion. Previous studies, using in situ hybridization, revealed that the predominant proteinase expressed by the implanting mouse embryo is matrix metalloproteinase-9 (MMP-9), and the major tissue inhibitor of metalloproteinases (TIMP) produced in murine endometrium during this period is TIMP-3. This study was performed to evaluate the expression of TIMP-3 mRNA during periimplantation period in the hyperstimulated murine endometrum. Design: Laboratory research. Materials/Methods: Quantitative, competitive-reverse transcription-polymerase chain reaction (QC-RT-PCR) was performed to evaluate the expression of TIMP-3 mRNA during periimplantation period in the hyperstimulated murine endometrum from pregnant uteri of gestation day (g.d.) 6.0 and 7.0 after injection of pregnant mare serum gonadotropin (PMSG) 5 IU and 10 IU. TIMP-3 mRNA expression was corrected by the simultaneous transcript level of matrix metalloproteinases-9 (MMP-9) which is mainly expressed by mouse embryos, since TIMP-3 is expressed mainly in the murine endometrium adjacent to implanting mouse embryos. Results: In both PMSG 5 IU and 10 IU groups, the expression of TIMP-3 mRNA showed an increase while MMP did not show a significant difference compared to control group (natural coitus without PMSG injection), on g.d. 6.0 and 7.0. Corrected TIMP-3 mRNA expression for MMP-9 also showed a high level of transcription in murine endometrium in the superovulation groups. Conclusions: This study suggests that ovarian hyperstimulation by gonadotropin, which produces many oocytes and embryos, may have a detrimental effect on embryonic implantation via its relevant endometrial remodeling process by the increase in expression of TIMP-3 in murine endometrium. Supported by: None.

Stage of the estrous cycle at the time of pregnant mare's serum gonadotropin injection affects pre-implantation embryo development in vitro in the mouse.

The present study aims to analyze in the mouse the effect of the stage of the estrous cycle at the time of pregnant mare's serum gonadotropin (PMSG) injection on fertilization of ovulated cumulus-enclosed oocytes and later embryo development in vitro to the blastocyst stage. Quality of blastocysts was evaluated by staining and counting of total number of nuclei, mitotic index, percentage of apoptotic nuclei, and cell allocation to the inner cell mass (ICM) and trophectoderm (TE) lineage. Superovulation of hybrid (C57Bl/6JIco female x CBA/JIco male) female mice of 4-6 weeks of age was induced by a priming injection of PMSG at different stages of the estrous cycle followed after a 48-hr interval by human chrorionic gonadotropin. Our data indicate that injection of PMSG at the estrus phase gives the best outcome whereas injection of PMSG at the diestrus-1 or diestrus-2 phase provides the worst results. In fact, (1) total number of oocytes ovulated, number of ovulated oocytes enclosed by cumulus cells, and number of TE cells in day-5 blastocysts were significantly lower in diestrus-1 females than in estrus, diestrus-2 and proestrus mice; (2) percentage of day-5 blastocysts and total number of cells in day-5 blastocysts were lower in diestrus-1 and diestrus-2 females than in estrus and proestrus mice; and (3) percentage of apoptotic nuclei in day-5 blastocysts was lower in estrus mice than in diestrus-1, diestrus-2, or proestrus females. These data endorse previous studies suggesting that administration of gonadotropins in mice should be synchronized with the innate estrous cycle of females.

Effect of feeding and body condition score on multiple ovulation and embryo production in zebu cows.

The aim of this study was to standardize the feeding regimen and the body condition score (BCS) for maximum superovulatory responses in indigenous zebu cows. Ten regularly cycling 5-8-year-old dry cows, weighing 176--260 kg with BCS 2.5--4.5 were divided into two equal groups at random. The groups were maintained on either a good-nutrition or a high-nutrition diet. The feedstuffs were analysed by proximate feed analysis and the metabolizable energy content was estimated. After 3 months feeding, individual cows were injected (i.m) with 1500 IU pregnant mare's serum gonadotrophin (PMSG) at day 10 or day 11 of the oestrous cycle (day of oestrus = day 0). Alfaprostol (6 mg) was injected (i.m) 48 h after the injection of PMSG to induce oestrus. At day 6 or day 7 (day of insemination = day 1), the contents of individual uterine horns were flushed with 150-200 ml of phosphate-buffered saline + 0.2% bovine serum albumin using a two-way Foley catheter. The embryos were identified, evaluated and graded as excellent, good, fair or poor under a stereomicroscope. For the good- and high-nutrition diets, the daily intake of green grass, straw, concentrate, dry matter, crude protein and estimated metabolizable energy by individual cows were 5 and 6 kg, 3 and 3 kg, 1.5 and 3.5 kg, 4.87 and 6.82 kg, 0.39 and 0.74 kg, and 39.60 and 59.12 MJ, respectively. The protein content was 8 and 11% in the good- and high-nutrition diets, respectively. The two groups of cows on different nutritional diets differed significantly with regard to body weight, body condition score and number of palpated corpora lutea (p < 0.01). For cows on the good-nutrition diet, the median number of recovered embryos and transferable quality embryos were three and two, respectively. The recovery rate of embryos was 79.30% of palpated corpora lutea. Cows on the high-nutrition diet did not yield any embryos. The indigenous zebu cows fed on the good-nutrition diet with BCS 2.5-3 were considered suitable for the induction of superovulation, the cows on the high-nutrition diet with BCS 4-4.5 were unsatisfactory and were more prone to cyst formation in the ovaries.

Stage of the estrous cycle at the time of pregnant mare's serum gonadotropin injection affects the quality of ovulated oocytes in the mouse.

The present study aims to analyze the effect of the stage of the estrous cycle at the time of pregnant mare's serum gonadotropin (PMSG) injection on number and quality of mouse oocytes retrieved from oviducts after exogenous ovarian stimulation. Cellular and morphological traits of ovulated oocytes from hybrid (C57Bl/6JIco female X CBA/JIco male) female mice of 12, 40-42, 50-52 or 57-62 weeks of age were analyzed. Superovulation was induced by a priming injection of PMSG at different stages of the estrous cycle followed after a 48-hr interval by human chrorionic gonadotropin. Injection of PMSG at diestrus-1 was associated with: (1) increased percentage of cumulus-free oocytes; (2) raised total percentage of oocytes without polar body; (3) increased total percentage of oocytes with intracytoplasmic mitochondrial aggregates; (4) decreased percentage of oocytes with a normal distribution of chromosomes in the metaphase II plate; and (5) raised percentage of oocytes with chromosome scattering when compared to injection at estrus, diestrus-2, and proestrus stage. On the contrary, estrus females displayed the highest percentage of oocytes with a normal distribution of chromosomes in the metaphase II plate and the lowest percentage of oocytes denuded of cumulus cells, without polar body, with intracytoplasmic mitochondrial aggregates and/or with chromosome scattering. These data suggest that administration of gonadotropins in mice should be synchronized with the innate estrous cycle of females to optimize the quality of oocytes collected from oviducts.

Expression of interleukin-18 and its receptor in mouse ovary.

Interleukin-18 (IL-18) strongly induces interferon-gamma production and is produced not only by types of immune cells but also by types of non-immune cells. Ovulation is thought to be an inflammation-like reaction in which many pro-inflammatory cytokines are involved. We investigated whether IL-18 is involved in the functions of ovary. The 4-week-old immature female mice were examined for IL-18 and IL-18 receptor (IL-18R) expression on their ovaries under stimulation with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) by immunohistochemical staining, Western blotting and reverse transcript-polymerase chain reaction. The IL-18R was blocked by the injection of anti-IL-18R monoclonal antibody to immature mice during PMSG-hCG stimulation, and the number of ovulated ova was counted. The expression of both proteins and mRNA of IL-18 and IL-18R were very low in immature ovaries before stimulation, but after PMSG injection both IL-18 and IL-18R increased dramatically in theca cells and reached a maximum level at the peri-ovulatory phase then slightly lowered, but still kept a high level during the luteal phase in the corpus luteum. The treatment of IL-18R monoclonal antibody to the mice during ovarian stimulation reduced the number of ovulated ova and inhibited the expansion of cumulus cells surrounding the ovum. IL-18 and IL-18R play roles in various kinds of function of ovary.

Expression of Ovarian Prolactin Receptor in Relation to Hormonal Changes during Induction of Ovulation in the Rat

We examined the relationships between the expression of the short and long forms of the prolactin (PRL) receptor (PRLR) mRNA in the ovary and changes in the levels of serum hormones such as sex steroid hormones and PRL during induction of ovulation in the rat. The expression of both forms of PRLR mRNA in the ovary was examined by Northern blot analysis in immature female rats treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Ovarian tissues and blood samples were obtained before treatment, 24 and 48 h after PMSG injection and 4, 6, 8, 12, 24 and 48 h after hCG treatment. Serum levels of 17β-estradiol, progesterone and PRL were determined by radioimmunoassay. Serum levels of 17β-estradiol rapidly increased to a maximal level 48 h after PMSG injection and then rapidly declined until 4 h after hCG injection. Serum levels of progesterone gradually increased after PMSG treatment, markedly increased to 114.2 nmol/l 8 h after hCG treatment and remained high until 48 h after hCG treatment. The serum level of PRL peaked at 66.2 µg/l (p < 0.01) 48 h after PMSG injection, and a temporary decrease after hCG treatment was followed by a continuously high level from 8 to 48 h. The expression of the long form of PRLR mRNA increased significantly (p < 0.01) to 688% of the control level after PMSG treatment, while that of the short form increased to only 184% of the control level. The expression of the long form of PRLR-mRNA rapidly declined until 6 h and then gradually increased until 48 h after hCG treatment. On the other hand, the expression of the short form of PRLR mRNA decreased to a nadir 12 h after hCG injection and then increased significantly (p < 0.01) to 142% of the control level. Our results showed that the changes in the short and long forms of PRLR mRNA differed in a time-specific manner and that these two forms are involved in different functions in the rat ovary during induction of ovulation. It is thought that the long form of PRLR mRNA is involved in folliculogenesis, while the short form of PRLR mRNA may play an important role in the formation and maintenance of the corpus luteum in the rat ovulatory cycle.