Long-term imatinib diminishes ovarian reserve and impacts embryo quality.

PURPOSE: Tyrosine kinase inhibitors (TKIs) such as imatinib are commonly used chemotherapeutics, but the effects of long-term treatments on reproductive outlook for cancer survivors are unknown. The purpose of this study was to examine the effects of long-term imatinib treatments on follicle development and embryo quality. Since prospective studies are not possible in healthy humans, we have incorporated a commonly used mouse model. METHODS: Adult female mice were treated with daily IP injections of imatinib for 4-6 weeks. Liquid chromatography-mass spectrometry was used to measure imatinib in serum and ovarian tissues. At the end of treatments, females were superovulated and mated to yield fertilized embryos. Oocytes and embryos were collected from oviducts, assessed for development by microscopy, and fertilized embryos were cultured in vitro. Blastocysts were fixed and stained for differential cell counts. RESULTS: Long-term imatinib treatments caused a shift in follicle development, with imatinib-treated females having fewer primordial follicles, but an increase in primary and secondary follicles (P < 0.05). There was no effect on ovulation or fertilization rates. However, blastocysts from imatinib-treated females had fewer total cells (P < 0.05) and a significant shift from inner cell mass to increased trophectoderm cells. CONCLUSION: This pilot study indicates that long-term TKI treatments may have significant impact on ovarian reserve and embryo developmental capacity. More studies are needed in other model systems to determine the long-term impact of TKIs in patients. Knowing the potential effects of chemotherapeutics on reproductive outlook is critical for quality of life and more research is needed.

Superovulation alters the expression of endometrial genes critical to tissue remodeling and placentation.

PURPOSE: Epidemiologic data suggest that in vitro fertilization (IVF) is associated with an increased risk of disorders of placentation including preeclampsia and fetal growth restriction. Specifically, studies have demonstrated that singleton pregnancies conceived following a fresh embryo transfer are at an increased risk of delivering an infant with low birth weight compared to those conceived following a frozen embryo transfer. The mechanism responsible for this association remains unclear. Procedures utilized in IVF have also been linked with epigenetic changes and gene expression changes in both fetal and maternal tissues. Data suggest that modifications in the maternal endometrium can lead to disordered trophoblast invasion and placentation. This study examines the effect of ovarian stimulation on endometrial gene expression and DNA methylation during the window of implantation to examine potential pathways playing a role in the adverse outcomes associated with IVF. METHODS: Endometrial biopsies were obtained from oocyte donors and age-matched naturally cycling women 11 days following oocyte retrieval in donors or 12 days following luteinizing hormone (LH) surge in naturally cycling women. Global gene expression was analyzed via Affymetrix Human Gene 1.1 ST array and confirmed with RT-qPCR. DNA methylation was assessed with the Infinium DNA methylation 450 K BeadChip. RESULTS: Analysis of endometrial gene expression from 23 women (11 oocyte donors and 12 controls) demonstrated 165 genes with a greater than twofold change in expression between donors and controls. While there were 785 genes with significant differential methylation in the endometrium of donors when compared with control subjects, none of the genes with altered expression showed significant changes in DNA methylation. Analysis of the differentially expressed genes showed enrichment for genes involved in endometrial remodeling including PLAT, HSPE2, MMP2, and TIMP1. Validation studies using RT-qPCR found a 73% reduction in expression of heparanase 2 (HSPE2) an enzyme associated with both angiogenesis and cell invasion, a greater than twofold increase in tissue-type plasminogen activator (PLAT), a serine protease participating in matrix degradation, and a 70% increase in MMP2, a gelatinase involved in collagen and fibronectin breakdown. CONCLUSIONS: Superovulation alters expression of genes critical to endometrial remodeling during early implantation. Such changes could lead to altered trophoblast migration and impaired endovascular invasion. These findings offer a potential mechanism for the adverse perinatal outcomes observed following embryo transfer during fresh IVF cycles.

Effect of superstimulation on the expression of microRNAs and genes involved in steroidogenesis and ovulation in Nelore cows.

To better understand the impact of ovarian superstimulation on bovine follicular microenvironment, Nelore cows (Bos taurus indicus) were subjected to ovarian superstimulation with follicle stimulating hormone (FSH, n = 10; P-36 protocol) or FSH combined with eCG (n = 10; P-36/eCG protocol). Follicular fluid was analyzed for cholesterol concentration. Granulosa cells were analyzed by RT-qPCR to assess the expression of genes involved in steroidogenic and ovulatory and expression of microRNAs involved in final follicular development and luteinizing hormone/choriogonadotropin receptor (LHCGR) expression. Plasma concentration of estradiol was also measured. Follicular fluid from the P-36 group showed higher concentration of cholesterol than that of control (non-superstimulated) cows. Plasma concentration of estradiol was higher in the P-36/eCG group. Abundance of STAR and FSHR mRNAs were lower in granulosa cells from the P-36/eCG group. In contrast, LHCGR mRNA abundance was higher in superstimulated granulosa cells from the P-36 group and showed a pattern opposite to that of miR-222 expression. Ovarian superstimulation did not affect the expression of other markers (mmu-miR-202-5p, has-miR-873, has-miR-144, and their target genes, CREB, TGFBR2, and ATG7) of antral follicle development. However, the mRNA expression of VEGF pathway components was modulated by P-36 treatment. Taken together, these results demonstrate that superstimulatory protocols modify steroidogenic capacity, increase plasma estradiol, and regulate the abundance of VEGF system, LHCGR mRNA and suppress the expression of miR-222 in bovine granulosa cells.

Polymorphism of the inhibin ßA gene and its relationship with superovulation traits in Chinese Holstein cows.

Inhibin is a major regulator of secretion of follicle-stimulating hormone, which is involved in follicular development and regulation of steroidogenesis in females. The objectives of this study were to detect polymorphisms of the bovine inhibin beta-A subunit (INHßA) gene and to evaluate its associations with superovulatory responses in 171 Chinese Holstein cows treated for superovulation. Polymerase chain reaction-restricted fragment length polymorphism revealed a C>T transition determining the StyI polymorphism at position 7639 in intron I of the bovine INHßA gene, and three genotypes (CC, CT, and TT) were detected. The frequencies of the three genotypes showed a tendency for CT > TT > CC, and this polymorphism was in Hardy-Weinberg equilibrium. Statistical analysis revealed no significant differences of least square means for superovulation traits among the three genotypes (P > 0.05). These results demonstrate, for the first time, that the detected loci of the INHßA gene have no significant effects on superovulation performance in Chinese Holstein cows.

Effects in cattle of genetic variation within the IGF1R gene on the superovulation performance and pregnancy rates after embryo transfer.

The insulin-like growth factor 1 receptor (IGF1R) is a membrane glycoprotein mediating most biological actions of IGF1 and IGF2, and has an important effect on ovulation, pre-implantation embryo development and pregnancy rate. The objectives of this study were to detect IGF1R gene polymorphisms of cattle and analyze the relationship with superovulation performance and pregnancy rates after embryo transfer (ET), as well as the hormone concentrations at the day of ET. One reported SNP of IGF1R G404T and a novel SNP of IGF1R G399A were analyzed in 170 Chinese Holstein donor cows and 118 Luxi recipients cattle. Statistical analysis revealed that the G404T mutation was associated (p=0.019) with increased ovulation rate and females with this mutation had enhanced performance in producing transferable embryos. For the polymorphic locus G399A, recipients with g.399 GG and g.399 GA genotypes had greater pregnancy rates after ET than that of g.399 AA genotype. Furthermore, the same tendency was observed that the genotype groups with greater pregnancy rates had greater progesterone and lesser estrogen concentrations, but these did not reach statistical significance. Results of the present study showed, for the first time, that the polymorphism in IGF1R is associated with superovulation traits, and indicated that the IGFIR gene can be used as a potential marker for donor selection.

The imitation switch ATPase Snf2l is required for superovulation and regulates Fgl2 in differentiating mouse granulosa cells.

Imitation switch (ISWI) proteins are catalytic subunits of chromatin remodeling complexes that alter nucleosome positioning by hydrolyzing ATP to regulate access to DNA. In mice, there are two paralogs, SNF2-homolog (SNF2H) and SNF2-like (SNF2L), which participate in different complexes and have contrasting patterns of expression. Here we investigate the role of SNF2L in ovaries by characterizing a mouse bearing an inactivating deletion of exon 6 that disrupts the ATPase domain. Snf2l mutant mice produce significantly fewer eggs than control mice when superovulated. Gonadotropin stimulation leads to a significant deficit in secondary follicles and an increase in abnormal antral follicles. Mutant females also failed to induce fibrinogen-like 2 (Fgl2) in response to human chorionic gonadotropin (hCG) stimulation, while overexpression of SNF2L was sufficient to drive its expression in granulosa cells. SNF2L was also shown to directly interact with the nuclear receptor co-activator flightless I (FLI-I) as shown by immunoprecipitation. These results begin to establish a role for SNF2L in the precise coordination of gene expression in granulosa cells during folliculogenesis and its broader implications in fertility.

Dual effects of superovulation: loss of maternal and paternal imprinted methylation in a dose-dependent manner.

Superovulation or ovarian stimulation is currently an indispensable assisted reproductive technology (ART) for human subfertility/infertility treatment. Recently, increased frequencies of imprinting disorders have been correlated with ARTs. Significantly, for Angelman and Beckwith-Wiedemann Syndromes, patients have been identified where ovarian stimulation was the only procedure used by the couple undergoing ART. In many cases, increased risk of genomic imprinting disorders has been attributed to superovulation in combination with inherent subfertility. To distinguish between these contributing factors, carefully controlled experiments are required on spontaneously ovulated, in vivo-fertilized oocytes and their induced-ovulated counterparts, thereby minimizing effects of in vitro manipulations. To this end, effects of superovulation on genomic imprinting were evaluated in a mouse model, where subfertility is not a confounding issue. This work represents the first comprehensive examination of the overall effects of superovulation on imprinted DNA methylation for four imprinted genes in individual blastocyst stage embryos. We demonstrate that superovulation perturbed genomic imprinting of both maternally and paternally expressed genes; loss of Snrpn, Peg3 and Kcnq1ot1 and gain of H19 imprinted methylation were observed. This perturbation was dose-dependent, with aberrant imprinted methylation more frequent at the high hormone dosage. Superovulation is thought to primarily affect oocyte development; thus, effects were expected to be limited to maternal alleles. Our study revealed that maternal as well as paternal H19 methylation was perturbed by superovulation. We postulate that superovulation has dual effects during oogenesis, disrupting acquisition of imprints in growing oocytes, as well as maternal-effect gene products subsequently required for imprint maintenance during pre-implantation development.

Gonadotropin stimulation increases the expression of angiotensin-(1--7) and MAS receptor in the rat ovary.

We have previously shown the presence of immunoreactive angiotensin-(1-7) [Ang-(1-7)] in rat ovary homogenate and its stimulatory effect on estradiol and progesterone production in vitro. In the current study, we investigated the presence and cellular distribution of Ang-(1-7) and the Mas receptor, the expression of Mas and angiotensin-converting enzyme 2 (ACE2) messenger RNA (mRNA), and the enzymatic activity in the rat ovary following gonadotropin stimulation in vivo. Immature female Wistar rats (25 days old) were injected subcutaneously (SC) with equine chorionic gonadotropin (eCG, 20 IU in 0.2 mL) or vehicle 48 hours before euthanasia. Tissue distributions of Ang-(1-7), Mas receptor, and ACE2 were evaluated by immunohistochemistry, along with angiotensin II (Ang II) localization, while the mRNA expression levels of Mas receptor and ACE2 were evaluated by real-time polymerase chain reaction (PCR). In addition, we determined the activity of neutral endopeptidase (NEP), prolyl endopeptidase (PEP), and ACE by fluorometric assays. After eCG treatment, we found strong immunoreactivity for Ang-(1-7) and Mas primarily in the theca-interstitial cells, while Ang II appeared in the granulosa but not in the thecal layer. Equine chorionic gonadotropin treatment increased Mas and ACE2 mRNA expression compared with control animals (3.3- and 2.1-fold increase, respectively; P < .05). Angiotensin-converting enzyme and NEP activities were lower, while PEP activity was higher in the eCG-treated rats (P < .05). These data show gonadotropin-induced changes in the ovarian expression of Ang-(1-7), Mas receptor, and ACE2. These findings suggest that the renin-angiotensin system (RAS) branch formed by ACE2/Ang-(1-7)/Mas, fully expressed in the rat ovary and regulated by gonadotropic hormones, could play a role in the ovarian physiology.

Characterization of the ovarian and reproductive abnormalities in prepubertal and adult estrogen non-responsive estrogen receptor alpha knock-in (ENERKI) mice.

Estrogen non-responsive estrogen receptor alpha (ERalpha) knock-in (ENERKI) mice have a mutation (glycine 525 to leucine, G525L) in the ligand-binding domain of ERalpha. The mutant ERalpha protein has a significantly lower affinity and response to endogenous estrogens, while not altering growth factor activated ligand-independent pathways. ENERKI females demonstrated signs of early follicle development as determined by a significant increase in antral follicle formation by 20 days of age. Adult ENERKI females were infertile, had hemorrhagic ovarian follicular cysts, and failed to develop corpora lutea in response to a superovulation regimen. These results illustrate the importance of ERalpha ligand-induced signaling for ovarian development and for estrogen feedback on the hypothalamus and pituitary. Although ERalpha ligand-induced signaling by endogenous estrogens is lost in ENERKI females, the ERalpha selective agonist propyl pyrazole triol (PPT), a synthetic nonsteroidal compound, is still able to activate G525L ERalphain vivo to increase uterine weight. To test whether PPT could restore ligand-dependent receptor activation, ENERKI females were treated with PPT and evaluated for spontaneous ovulation, ovarian hemorrhagic cysts, and LH serum levels. Daily PPT treatments beginning on day 4 of life prevented formation of ovarian hemorrhagic cysts in adult ENERKI animals. In accordance with this result, preputial gland weight and LH levels were also lowered in these animals, indicating PPT treatments most likely led to restoration of ERalpha negative feedback of the hypothalamic-pituitary axis.

Superovulation in mice alters the methylation pattern of imprinted genes in the sperm of the offspring.

Some steps of the assisted reproduction techniques, such as superovulation, may interfere with imprinting reprogramming. In the present study, superovulation was induced in the mouse and its possible effects on the differentially methylated domains of 2 paternally (H19 and Gtl2) and 3 maternally (Peg1, Snrpn and Peg3) imprinted genes were tested in the male offspring over 2 generations. The CpGs methylation status was analyzed by pyro- and bisulfite sequencing. In liver, skeletal muscle and tail, no effect of superovulation could be observed. In the sperm, however, a significant 6% decrease in the number of methylated CpGs of H19 and significant 2.8- and 7.0-fold increases in those of Peg1 and Snrpn, respectively were observed following superovulation. The changes were still present in the H19 and Snrpn genes of the second generation offspring. This suggests that superovulation in the mother transgenerationally affects the offspring sperm methylation pattern.

ERK signaling in the pituitary is required for female but not male fertility.

Males and females require different patterns of pituitary gonadotropin secretion for fertility. The mechanisms underlying these gender-specific profiles of pituitary hormone production are unknown; however, they are fundamental to understanding the sexually dimorphic control of reproductive function at the molecular level. Several studies suggest that ERK1 and -2 are essential modulators of hypothalamic GnRH-mediated regulation of pituitary gonadotropin production and fertility. To test this hypothesis, we generated mice with a pituitary-specific depletion of ERK1 and 2 and examined a range of physiological parameters including fertility. We find that ERK signaling is required in females for ovulation and fertility, whereas male reproductive function is unaffected by this signaling deficiency. The effects of ERK pathway ablation on LH biosynthesis underlie this gender-specific phenotype, and the molecular mechanism involves a requirement for ERK-dependent up-regulation of the transcription factor Egr1, which is necessary for LHbeta expression. Together, these findings represent a significant advance in elucidating the molecular basis of gender-specific regulation of the hypothalamic-pituitary-gonadal axis and sexually dimorphic control of fertility.

Comparação de diferentes técnicas de sincronização da emergência da onda folicular visando a superovulação em bovinos / Comparative technics of follicular wave emergence synchronization in the cattle superovulation

Foram avaliadas quatro técnicas de sincronização da onda folicular em protocolos de superovulação. Para tal, foram utilizadas 112 vacas doadoras, das raças Simental, Limousin e Red Angus, com escore corporal médio de 3,0. Os animais foram divididos aleatoriamente em cinco grupos experimentais de acordo com o método de sincronização da emergência da onda folicular. Foram realizadas 30 superovulações em cada grupo, considerando os seguintes protocolos: GI - grupo controle – animais superovulados entre o 8o e o 12o dia do ciclo estral (dia zero = estro); GII – animais que sofreram punção folicular no 9o dia (dia 0 = estro) e início do tratamento superovulatório no 11o dia; GIII – animais que sofreram punção folicular em fase não conhecida do ciclo estral, associada ao uso de um dispositivo intravaginal contendo progesterona (P4) e tratamento superovulatório iniciado 48h após a punção, GIV – animais que utilizaram implante intravaginal de progesterona colocadoem fase aleatória do ciclo estral, mantido por nove dias, associado à administração de 50 mg de P4 e de 2mg de benzoato de estradiol, sendo o tratamento superovulatório iniciado cinco dias após a colocação do dispositivo e GV – animais que receberam implante intravaginal de P4 colocado em fase aleatória do ciclo estral e mantido por oito dias, associado à administração de 50mg de P4 e 2mg de 17â-estradiol, sendo o tratamento superovulatório iniciado quatro dias após a colocação do dispositivo. Nos grupos I, II, III, IV e V o total de estruturas coletadas e de embriões viáveis foram, respectivamente (13,53±9,23 vs 13,87 ± 7,85 vs 18,70 ± 10,88 vs 9,03 ± 4,97 vs 13,60 ± 8,39) e (8,43±5,68 vs 8,27 ± 7,06 vs 10,47 ± 8,19 vs 5,37 ± 2,92 vs 7,23 ± 5,30). Os resultados observados no GIII foram superiores ao GI, GII, GIV e GV (P 0,05), enquanto o desempenho de GIV foi inferior (P<0,05). Os resultados permitem concluir que é possível sincronizar a emergência da onda folicular de vacas doadoras, com início da superovulação em qualquer momento do ciclo estral, e que o tratamento progestágeno associado à punção folicular oferece os melhores resultados.

We evaluated four different techniques of follicular wave synchronization by comparing total number of structures recovery, number of viable and degenerated embryos, number of oocytes recovery and cost of uterine flushing. One hundred twelve Simental, Limousin and Red Angus donators were randomly allocated in five treatment groups according to protocol used for follicular wave emergence synchronization. Thirty superovulations were performed in each group, the programs were: G1- Control, superovulation treatment between days 8 and 12 of the estrous cycle; G2- follicle aspiration on day 9 of the estrous cycle; G3- follicle aspiration plus intravaginal progesterone implant; G4- intravaginal progesterone implant plus estradiol benzoate and G5- intravaginal progesterone implant plus estradiol-17â. Artificial insemination was performed twice, 12 and 24 hours after detection of behavioral estrus. Embryo collection was performed on day 7 after inseminations. Total number of recovered structures (18,70 ± 10,88 vs 13,53 ± 9,23) and viable embryos (10,47 ± 8,19 vs 8,43 ± 5,68) were higher (P0,05) were detected amongst groups 2, 5 and 1, while performance of G4 was the lowest (P<0,05). Results demonstrate to be possible synchronize follicular wave emergence by initiating superstimulation at any time of the estrous cycle. Additionally we verified that the program using progesterone associated to follicular ablation had the best results in synchronizing follicular wave emergence aiming superstimulation in cattle.

Gene injections of vascular endothelial growth factor and growth differentiation factor-9 stimulate ovarian follicular development in immature female rats.

OBJECTIVE: To examine the effect of vascular endothelial growth factor (VEGF) and growth differentiation factor-9 (GDF-9) on follicular development of the ovaries in immature female rats. DESIGN: Superovulation and gene injection. SETTING: Animal reproduction laboratory in Tohoku University, Sendai, Japan. ANIMAL(S): Wister-Imamichi female rats. INTERVENTION(S): The ovulated oocytes from rats with injected VEGF and GDF-9 gene fragments were counted, and the ovaries removed from those rats were used in the histologic observation. MAIN OUTCOME MEASURE(S): Follicular dynamics and angiogenesis after VEGF and GDF-9 gene fragments injection. RESULT(S): A single injection of the VEGF gene led to the production of a large number of oocytes (approximately 110 oocytes) from an individual animal that was injected with the gene at 21 days after birth, and after mating most of the oocytes were fertilized. Direct ovarian injection of GDF-9 stimulated the development of medium-sized antral follicles. The number of ovulated oocytes after injection of the VEGF plus GDF-9 gene fragments was the same as with a single injection of the VEGF gene. CONCLUSION(S): A single injection of the VEGF or GDF-9 gene stimulated follicular development, and injection of both genes did increase the number of ovulated oocytes from individual animals. An exogenous gene fragments injection promoted the maximum potential of ovarian function in immature female rats.

Conditional induction of ovulation in mice.

Follicle-stimulating hormone controls the maturation of mammalian ovarian follicles. In excess, it can increase ovulation (egg production). Reported here is a transgenic doxycycline-activated switch, tested in mice, that produced more FSHB subunit (therefore more FSH) and increased ovulation by the simple feeding of doxycycline (Dox). The transgenic switch was expressed selectively in pituitary gonadotropes and was designed to enhance normal expression of FSH when exposed to Dox, but to be regulated by all the hormones that normally control FSH production in vivo. Feeding maximally effective levels of Dox increased overall mRNA for FSHB and serum FSH by over half in males, and Dox treatment more than doubled the normal ovulation rate of female mice for up to 10 reproductive cycles. Lower levels of Dox increased the number of developing embryos by 30%. Ovarian structure and function appeared normal. In summary, gene switch technology and normal FSH regulation were combined to effectively enhance ovulation in mice. Theoretically, the same strategy can be used with any genetic switch to increase ovulation (or any highly conserved physiology) in any mammal.

Transcripts of neurokinin B and neurokinin 3 receptor in superovulated rat ovaries and increased number of corpora lutea as a non-specific effect of intraperitoneal agonist application.

Neurokinin B (NKB), a member of the tachykinin family, and its neurokinin 3 receptor (NK3-R) are preferentially found in the central nervous system. Others have recently reported on mRNA from this ligand-receptor system in the uterus and on NK3-R expression increasing with age. NKB and NK3-R mRNAs have also been noted in cumulus cells and oocytes from superovulated rats. Intact ovaries before and after puberty have not been studied. In this study, we stimulated 29-day-old rats by s.c. injections with gonadotropins for estrous cycle synchronization in order to elucidate the NKB-NK3-R system's expression and function in the ovary. Simultaneously, NaCl, the NK3-R agonist (Pro(7))-NKB, the antagonist SB 218795, or thiorphan, a neutral endopeptidase inhibitor of tachykinin degradation, were injected intraperitoneally (i.p.) for 3 1/2 consecutive days. First, we demonstrated NKB and NK3-R transcripts in one rat ovary by RT-PCR. No significant mRNA differences were noted between immature ovaries and superovulated ovaries in any of the i.p. applications. Second, the possible role of NK3-R on the ovulatory process was verified by counting corpora lutea (CL) and CL cysts in serial sections of the other ovary derived from the four different groups and embedded in paraffin wax. CL and CL cysts were noted in greater numbers in the pharmacologically treated groups than in the saline-treated group. To validate possible drug effects on the peritoneum, we additionally studied pieces of the omentum majus and retroperitoneal fat tissue. Both tissues were heavily infiltrated by granulocytes similar to a non-specific inflammatory response. The saline-treated group as well as the pharmacologically treated groups appeared to develop this unexpected side effect to a similar degree. We conclude that transcripts of NKB and NK3-R are present before and after puberty in the rat ovary and appear to be expressed at similar levels which may indicate a role for the NKB-NK3-R system in follicle growth. The effect of increased CL formation after application of the NK3-R agonist i.p. is related to a non-specific response.

Increased frequency of sister-chromatid exchange and altered alkaline comet assay scores in superovulation cycles for unexplained infertility.

OBJECTIVE: Sister-chromatid exchange analysis (SCE) and alkaline comet assay measure and analyze DNA damage in mammalian cells. This study assesses whether ovulation induction agents increase DNA damage in the lymphocytes of infertile women undergoing superovulation. STUDY DESIGN: Prospective case control study in a university based hospital. Baseline and hCG day peripheral blood samples were withdrawn from 20 women with undergoing superovulation for unexplained infertility and baseline and luteinizing hormone (LH) peak day samples were also withdrawn from another 20 infertile women with unexplained infertility. RESULTS: There was increased SCE frequency and DNA damage determined by comet assay on the hCG day compared to the basal state. The SCE increase was correlated with the hCG day estradiol (E2) but not with ampoules of follicular stimulating hormone (FSH). The SCE frequency was also increased naturally ovulating women; however this was significantly less than that in FSH receiving women. CONCLUSION: Increased DNA damage may indicate for increased potential for malignancies after superovulation.

Genetic influences on reproduction of female red deer (Cervus elaphus) (2) seasonal and genetic effects on the superovulatory response to exogenous FSH.

This study evaluated the influences of seasons and genotype on the superovulatory response to a standardised oFSH regimen in red deer (Cervus elaphus scoticus) and its hybrids with either wapiti (C.e. nelsoni) or Père David's (PD) deer (Elaphurus davidianus). Adult red deer (n=9), F(1) hybrid wapiti x red deer (n=6), and maternal backcross hybrid PD x red deer (i.e., 14 PD hybrid; n=9) were kept together in the presence of a vasectomised stag for 13 months. At 6 weekly intervals, all hinds received a standardised treatment regimen used routinely to induce a superovulatory response in red deer hinds, with 10 consecutive treatments spanning an entire year. This involved synchronisation with intravaginal progesterone devices and delivery of multiple injections of oFSH (equivalent to 72 units NIH-FSH-S(1)). Laparoscopy to assess ovarian response was performed 6-7 days after the removal of the devices. Both season and genotype had significant effects on ovulation rate (OR) and total follicular stimulation (TFS) (P<0.05). For all the three genotypes, ovarian responses were highest from March to November (breeding season) and lowest in the period from December to January, inclusive. Mean OR for red deer hinds ranged from 3.7 to 1.8 during the breeding season, with no observable trend. All red deer hinds were anovulatory during December and January. A similar pattern occurred for 14 PD hybrids, although mean OR during the breeding seasons were twofold lower than for the red deer. For F(1) wapiti hybrids, the first two treatments in March and April resulted in the highest mean OR observed (15.6 and 11.7, respectively). Thereafter, mean values ranged between 6.3 and 4.7 for the remainder of the breeding season. Furthermore, mean OR of 3.0 and 0.5 were recorded in December and January, respectively. For the red deer and F(1) wapiti hybrids, between-hind variation in OR was not randomly distributed across the treatment dates, indicating that the individuals varied significantly in their ability to respond to oFSH, at least within a given season.In conclusion, the study has shown that relative to red deer, F(1) wapiti hybrid hinds exhibit a higher sensitivity to oFSH, whereas 14 PD hybrid hinds have a lower sensitivity. However, individual variation within genotype was very marked. A seasonal effect was apparent for all genotypes, although some F(1) wapiti hybrid hinds exhibited ovulatory responses throughout the year.

Prolongation of ovarian lifespan into advanced chronological age by Bax-deficiency.

Female mammals are endowed with a finite number of oocytes at birth, each enclosed by a single layer of somatic (granulosa) cells in a primordial follicle. The fate of most follicles is atretic degeneration, a process that culminates in near exhaustion of the oocyte reserve at approximately the fifth decade of life in women, leading to menopause. Apoptosis has a fundamental role in follicular atresia, and recent studies have shown that Bax, which is expressed in both granulosa cells and oocytes, may be central to ovarian cell death. Here we show that young adult female Bax-/- mice possess threefold more primordial follicles in their ovarian reserve than their wild-type sisters, and this surfeit of follicles is maintained in advanced chronological age, such that 20-22-month-old female Bax-/- mice possess hundreds of follicles at all developmental stages and exhibit ovarian steroid-driven uterine hypertrophy. These observations contrast with the ovarian and uterine atrophy seen in aged wild-type female mice. Aged female Bax-/- mice fail to become pregnant when housed with young adult males; however, metaphase II oocytes can be retrieved from, and corpora lutea form in, ovaries of aged Bax-/- females following superovulation with exogenous gonadotropins, and some oocytes are competent for in vitro fertilization and early embryogenesis. Therefore, ovarian lifespan can be extended by selectively disrupting Bax function, but other aspects of normal reproductive performance remain defective in aged Bax-/- female mice.

Respuesta superovulatoria en ganado Bos indicus y Bos taurus bajo condiciones tropicales, y efecto del desarrollo y calidad del embrión sobre el porcentaje de gestación / Superovulatory response in Bos indicus and Bos taurus cattle under tropical conditions, and effect of embryo development and quality on pregnancy rate

El presente estudio se realizó en condiciones de campo en 4 ranchos de la zona centro del estado de Yucatán. Los objetivos fueron, en primer lugar; evaluar la respuesta superovulatoria entre ganado Bos indicus y Bos taurus, y entre vacas y novillas; en segundo lugar, investigar el efecto del desarrollo y la calidad de los embriones transferidos sobre el porcentaje de gestación de las receptoras. Para esto, se superovularon 30 animales con hormona foliculoestimulante (FSH-P) y se transfirieron 88 embriones a un número igual de receptoras. El 83.3 por ciento de las donadoras (n=25), respondió satisfactoriamente al tratamiento superovulatorio, de éstas, se obtuvo en promedio 8.2 ñ 5.3 embriones recuperados por donadora, de los cuales 6.9 ñ 4.1 fueron embriones transferibles. Los animales Bos indicus presentaron un mayor promedio de embriones recuperados (p<0.10) y embriones transferibles por donadora (p>0.05) (11.6 ñ 7.9 y 7.8 ñ 4.2, respectivamente), que los animales Bos taurus (7.1 ñ3.6 y 6.6 ñ4.1, respectivamente). Al evaluar estos indicadores entre vacas y novillas, no se encontró diferencia en el promedio de los embriones recuperados por donadora (8.0 ñ 6.7 vs 8.3 ñ3.2, respectivamente: P>0.05); sin embargo, se observó cierta superioridad de las novillas en el promedio de embriones transferibles (7.9 ñ 3.8 vs 6.6 ñ 4.2, respectivamente), sin que esta diferencia sea significativa (P>0.05). El porcentaje global de gestación para las 88 receptoras fue de 51.1 por ciento (n=45), el porcentaje de gestación fue mayor cuando se transfirieron embriones en estadio de blastocisto (53.3 por ciento) que cuando se transfirieron en estadio de mórula (38.4 por ciento) (P>0.05), y también cuando se transfirieron embriones de calidad excelente (63.4 por ciento), comparados con los embriones de calidad buena, regular (44.8 por ciento y 44.4 por ciento, respectivamente: P>0.05) y mala (22.2 por ciento: P>0.05)