Pattern and frequency of nondisjunction in oocytes from the Djungarian hamster are determined by the stage of first meiotic spindle inhibition

In order to study the mechanisms of nondisjunction at meiosis I in oocytes gonadotropin-stimulated Djungarian hamsters were treated at two stages [4.5 and 6 h post human chorionic gonadotropin (HCG)] during the preovulatory period with 1000 mg/kg Carbendazim (MBC). The compound, known to bind fast but reversibly to mammalian tubulin, was chosen to investigate whether the stage at which spindle function is inhibited affects the pattern of nondisjunction. Ovulated oocytes were cytologically prepared and scored for hyperhaploidy, diploidy and presegregation. Application at an early spindle phase, 4.5 h post HCG, to females stimulated with a low gonadotropin dose [3 IU pregnant mares serum (PMS); 2 IU HCG] caused a high frequency of nondisjunction (40.6%) with a more or less nonspecific pattern of malsegregated bivalents. Treatment at a late stage of spindle function (6 h post HCG) resulted in a less frequent (22.5%) but highly preferential malsegregation of those A-D group bivalents thought earlier to be late segregators. On the other hand, oocytes from females primed with a high (10 IU PMS and HCG) gonadotropin dose, a treatment assumed to delay meiosis by approximately 1.5 h, responded to MBC treatment at the late stage (6 h) with a nonspecific pattern and a high frequency (71.2%) of nondisjunction. The latter result is comparable to that in which MBC was given at the early stage (4.5 h) and after a low gonadotropin dose. The high nondisjunction response additionally indicates that spindles in hypergonadotropic stimulated oocytes are more susceptible and/or that the concentration of the inhibitor is higher in such oocytes. Only few oocytes with presegregation (3.1%; 0.0%; 1.7%) and few diploid oocytes (3.3%; 1.5%; 3.2%) with complete inhibition of meiosis I were observed. We conclude, that in Djungarian hamsters (1) the segregation of bivalents at meiosis I is asynchronous with the large A-D bivalents segregating last, (2) the phase in which spindle function is inhibited determines the pattern of nondisjunction, and (3) the resumption of meiosis I — from dictyotene to metaphase II — does not follow a rigidly timed programme but depends on the conditions of follicular maturation.     

Effects of gonadotropins on oocyte maturation and progesterone production by porcine ovarian follicles cultured in vitro

Effects of gonadotropins on the maturation of isolated oocytes and production of progesterone by porcine ovarian follicles from gonadotropin treated gilts have been studied in vitro. The addition of gonadotropins (2 I. U./ml, PMSG, HGC or 2 mg/ml FSH) to the culture medium resulted in increasing the number (84 - 90 %) of isolated oocytes which reached metaphase II. Expansion of the whole cumulus mass was observed only in media containing PMSG, whereas FSH or HCG alone did not cause these marked changes in the cumulus cells. Denudation of the eggs prior to culture gave no significant differences in the maturation rates between oocytes cultured in media with or without gonadotropins. In vitro maturation of follicle-enclosed oocytes took place only in HCG treated animals. Removing the ovary at 15 or 60 minutes after intravenous HCG administration induced oocyte maturation only in 22% and 17% respectively. A sharp increase in the number of oocytes which resume meiosis during follicle culture was observed 4 hours after HCG injection (84 %) and all of the oocytes of the gilts ovariectomized at 8 hours after HCG injection matured during the culture period. The progesterone production of isolated follicles from control gilts (only PMSG injected) increased slowly during a 96-hour culture period (from 48 to 240 ng progesterone/follicle), whereas the secretion of progesterone was drastically increased after a 15 minute interval between HCG injection and ovariectomy (from 42 to 950 ng progesterone/follicle). Follicles removed 24 hours after HCG injection showed a further increase in steroid production (2000 ng progesterone/follicle) and consistently secreted large amounts of progesterone during the culture period.     

周龄和激素水平对长爪沙鼠超数排卵效果的影响

目的探讨不同周龄和激素水平对长爪沙鼠超数排卵效果的影响,以期确定长爪沙鼠最佳超排周龄和激素使用剂量。方法腹腔注射10 IU PMSG/HCG对4～18周龄8个年龄段的雌性长爪沙鼠进行超数排卵,末次注射16～17 h内对各组动物卵母细胞计数,确定最佳超排周龄后,对该年龄动物以5、10、15 IU3个剂量水平腹腔注射PMSG/HCG,观察各组动物的卵母细胞计数差异。结果与其它周龄组相比,6周龄组长爪沙鼠超数排卵后的卵母细胞数最多,各组间有统计学意义（P〈0.05）,而5、10、15IU等3个剂量组的超排效果也有一定的差异,10 IU组数量最高。结论对长爪沙鼠而言,采用10 IU激素注射和6周龄的动物进行超数排卵,获得的卵母细胞数量最多而且超排效果稳定性。     

Effects of a GnRH agonist on oocyte number and maturation in mice superovulated with eCG and hCG

The objective was to investigate the effects of a gonadotropin-releasing hormone agonist (GnRH) on ovulation rate and the number and maturation of oocytes in mice superovulated with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). Thirty 3-month-old BALB/C female mice (weight: 25-30 g) were assigned to three experimental groups: control, superovulated, and superovulated with GnRH pretreatment (n=10 per group). Control mice received an i.p. injection of 0.1 ml physiological saline solution. Superovulation was induced with 5 IU eCG (i.p.) and 5 IU hCG 48 h later. Mice in the superovulated with GnRH pretreatment group were given GnRH (20 mg/kg Fertirelin, i.m.), 24 h before superovulation. Thirteen hours after hCG administration, mice were sacrificed by cervical dislocation and blood samples were collected to determine serum progesterone concentration (by radioimmunoassay). Ovaries and oviducts were also harvested to enumerate corpora lutea and cumulus-enclosed oocytes. Progesterone concentrations were not significantly different among groups. The oocyte number and the maturation, ovulation rate, and the number of corpora lutea were higher in GnRH-treated mice than both controls and superovulated mice. In conclusion, GnRH given 24 h before superovulation with eCG-hCG increased the number and maturation of oocytes and the rate of ovulation in mice.     

C57BL/6J小鼠超数排卵的研究

目的　确定C57BL 6J小鼠超排的最佳激素剂量和最合适的注射间隔时间 ,提高超排率。方法　 40只C57BL 6J雌鼠随机分为四组 ,分别用 5IU或 10IU的PMSG和HCG ,间隔 48h或 72h注射 ,比较排出卵母细胞的数量。结果　 5IU +5IU剂量的PMSG和HCG、间隔 48h注射组超排效果最好 ;8～ 10周龄雌鼠较 6～ 8周龄雌鼠超排效果好。结论　C57BL 6J小鼠超排的最佳激素剂量为 5IUPMSG +5IUHCG ,最合适的注射间隔时间为 48h ,处于繁殖期的雌鼠超排效果好。     

Induction of superovulation and recovery of fertilized oocytes in prepubertal miniature pigs after treatment with PG600

The purpose of this study was to examine whether superovulation can be induced by hormonal treatment with PG600 (400 IU eCG and 200 IU hCG) at the prepubertal stage in miniature pigs. In Experiment 1, 14 prepubertal miniature pigs received 1, 1/2 or 1/4 vial of PG600, im on Day 0 (the first day of treatment). Presentation of estrus was monitored thereafter. On Days 10 to 13 (i.e., 6 to 8 d after estrus), the number of corpora lutea (CL) and residual follicles was counted by an exploratory laparotomy. Injection of 1/2 vial of PG600 effectively induced estrus and ovulation in the pigs. In Experiment 2, 15 prepubertal miniature pigs that received 1/2 vial of PG600 were artificially inseminated into the uterus by an exploratory laparotomy at 100 to 104 h after PG600 injection. Oocytes were recovered from the oviducts at 121 to 145 h after PG600 administration. The oocyte recovery rate was 66% (15 oocytes/pig, average), and 84% of these were at the 1-cell stage. In Experiment 3, 5 prepubertal miniature pigs that received 1/2 vial of PG600, followed by 100 IU hCG 70 h later, were artificially inseminated into the uterus. Oocytes were recovered synchronously at 120 to 122 h after PG600 treatment. The recovery rate was 80% (17 oocytes/pig, average) and 90% of the oocytes recovered were at the 1-cell stage. These results suggest that superovulation of prepubertal miniature pigs can be induced by 1/2 vial of PG600 injection, and by the combined treatment with PG600 and hCG injection, the fertilized ova can be synchronously recovered at around 120 h after PG600 injection. This procedure may provide a useful system for biomedical research using the miniature pigs, especially for producing transgenic animals for use in human disease models.     

SUPEROVULATION AND SUPERPREGNANCY IN THE GOLDEN HAMSTER*

Pregnant mare serum gonadotropin (PMSG) treatment given in the morning or afternoon on any day of the four-day estrous cycle and human chorionic gonado tropin (HCG) given two days later successfully induced superovulation in the golden hamster. The minimum interval between PMSG and HCG necessary to obtain consistent superovulation was approximately 44 hr. The lowest ovulation rate was obtained following PMSG treatment on the afternoon of day 4 despite the fact that this time coincides with the maximum endogenous FSH level, necessary for the maturation of the next crop of follicles destined to ovulate. Thirty-eight to one-hundred percent of superovulated females in four different treatment groups became superpregnant after natural mating. Some treated females exhibited two consecutive nights of estrus with ovulation apparently occurring during the second night. Superpregnant females delivered “super” size litters, up to 27 live-born pups. The ultimate litter size appeared to be established after day 3 and prior to day 8 of superpregnancy. A one-day extension of the normal 16-day gestation period was observed in 31% of superpregnancies. Unilateral pregnancies were observed at autopsy in 44% of treated females which received the high dose of PMSG (30 IU). The progeny of superovulated females reproduced normally at maturity. The results indicate that ova from superovulated female hamsters are capable of full normal development.     

Influence of ethanol on chromosome segregation during the first and second meiotic divisions in the mouse egg

This study was carried out to investigate the influence of ethanol on chromosome segregation during the first and second meiotic divisions. Female mice were given a single intragastric injection of a dilute solution of ethanol either just before or at various times after the HCG injection for inducing superovulation. The mice were mated, and the chromosome constitution of fertilized eggs was determined at the first cleavage mitosis. The technique employed allowed the male- and female-derived pronuclear sets to remain as two discrete groups. Exposure from 1.5 h before to 17 h after the HCG injection induced a high incidence of aneuploidy (15-25%) involving in over 90% of cases only one chromosome, so that either 19 or 21 instead of the normal complement of 20 chromosomes were present in one of the two sets (a previous study using a "marker" chromosome has demonstrated that the nondisjunction induced here invariably involves the female set). We suggest these findings draw attention to the susceptibility of chromosome segregation in female germ cells to interference by ethanol and that the mode of action is likely to be via interference with the normal functioning of the spindle apparatus. It is possible that interference with meiotic chromosome segregation by spindle-acting agents such as ethanol might account for a proportion of human spontaneous abortions with similar chromosomal defects where no other obvious cause is apparent.     

Studies on Recipient Rabbit Oocytes in Nuclear Transfer

Studies were carried out to find out the optimal parameters of enucleation and electroactivation ofrecipient rabbit oocyte for successful nuclear transfer,using the fluorescent stain,DAPI(4-6-diamidino-2-phenylindole),and electroactivation.According to the position of metaphasechromosomes in relation to the first polar body,the oocytes were classified into three types:1.thosewith chromosomes juxtaposed to the polar body;2.those with chromosomes in its adjacency; and3.those with chromosomes further removed.The relative proportions of each type appeared to varywith the time of maturation at which the oocytes were collected,with those of the later typesincreasing as the maturation process went further on.In addition,in-vitro cultivation ofelectroactivated oocytes gave the best results with oocytes that matured in-vito after injection ofovulating hormones(LH or HCG)and oocytes that were cultivated in-vitro for 17-19 hours.As aresult,it is recommended that oocytes be selected from those collected from the oviducts 13-15 hoursafter injection of LH or HCG,and electrofusion and electroactivation be done aftermicromanipulation and in-vito cultivation for 2-4 hours.By so doing,it is expected to achieve thehighest enucleation rate of oocytes and the highest fusion rate,the highest activation rate and thehighest development rate of the restructured embryos.     

APPEARANCE OF PRONUCLEUS-INDUCING ACTIVITY DURING HORMONALLY INDUCED MEIOTIC MATURATION IN TOAD OOCYTES*

Detergent-pretreated spermatozoa of the toad, Bufo bufo japonicus, transform into pronuclei when injected into progesterone-matured oocytes at 18 hr post-hormone treatment (PHT). These sperm, however, do not show any change when injected into the oocytes at the same age from which the germinal vesicle (GV) has been removed before the progesterone treatment. In an attempt to determine when and how the pronucleus-inducing activity (PIA) develops in hormonally induced maturation process, enucleated oocytes were injected with GV and sperm at various stages after the hormone treatment and electrically stimulated at 18 hr PHT. It was found that sperm pronuclei are induced only in those oocytes receiving GV before 14 hr PHT. The 1 hr pulse-treatment of maturing oocytes with cycloheximide between 8–18 hr PHT and the injection of sperm at 18 hr PHT revealed that PIA does not occur in the oocytes treated with the inhibitor during 10–14 hr PHT. Injection of α-amanitin into maturing oocytes had no effect in this respect. Determination of DNA synthetic activity in vitro of the oocyte extracts from various maturation stages showed that the net increase of the activity occurs before the formation of PIA. The activity of the cycloheximide-treated oocyte extracts utilizing native DNA did not correlate with the sensitivity of oocytes to the inhibitor with respect to PIA in situ. It is concluded that PIA develops, in association with the GV materials, by way of translational events at 10–14 hr PHT, being quiescent during later maturation stages, and commences to function as an activation response of oocytes at 18 hr PHT.     

Response of large oocytes of Xenopus laevis to progesterone in vitro in relation to oocyte size and time after previous HCG-induced ovulation.

The injection of Xenopus laevis females with human chorionic gonadotropin (HCG) leads to ovulation (and maturation) of oocytes whose diameters are 1.2 mm or larger. However, when Xenopus oocytes are removed from their follicular investments by manual dissection and exposed to the steroid, progesterone, in vitro, they exhibit maturation down to about 0.90 mm in diameter with the majority larger than 1.0 mm showing a positive response. Within each female the larger of the oocytes undergo maturation earlier than smaller ones.The response of oocytes also was shown to depend on the length of time since females were last stimulated to ovulate. Similar-sized oocytes from recently ovulated (stimulated) females matured much faster than those of untreated, unstimulated females. Indeed, even the smaller oocytes from stimulated females often matured before the largest oocytes of females without previous HCG injection.The experiments demonstrate that the physiological state of an oocyte cannot be accurately deduced solely from its size nor response to gonadotropins; unresponsiveness presumably being due to inability of follicular elements to respond to the trophic hormones or transfer the stimulus to the oocyte via the appropriate steroid.     

Different fates of oocytes with DNA double-strand breaks in vitro and in vivo

In female mice, despite the presence of slight DNA double-strand breaks (DSBs), fully grown oocytes are able to undergo meiosis resumption as indicated by germinal vesicle breakdown (GVBD); however, severe DNA DSBs do reduce and delay entry into M phase through activation of the DNA damage checkpoint. But little is known about the effect of severe DNA DSBs on the spindle assembly checkpoint (SAC) during oocyte maturation. We showed that nearly no first polar body (PB1) was extruded at 12 h of in vitro maturation (IVM) in severe DNA DSBs oocytes, and the limited number of oocytes with PB1 were actually at telophase. However, about 60% of the severe DNA DSBs oocytes which underwent GVBD at 2 h of IVM released a PB1 at 18 h of IVM and these oocytes did reach the second metaphase (MII) stage. Chromosome spread at MI and MII stages showed that chromosomes fragmented after GVBD in severe DNA DSBs oocytes. The delayed PB1 extrusion was due to the disrupted attachment of microtubules to kinetochores and activation of the SAC. At the same time, misaligned chromosome fragments became obvious at the first metaphase (MI) in severe DNA DSBs oocytes. These data implied that the inactivation of SAC during the metaphase-anaphase transition of first meiosis was independent of chromosome integrity. Next, we induced DNA DSBs in vivo, and found that the number of superovulated oocytes per mouse was significantly reduced; moreover, this treatment increased the percentage of apoptotic oocytes. These results suggest that DNA DSBs oocytes undergo apoptosis in vivo.     

Nuclear maturation in pig and rabbit oocytes after interspecific fusion

Porcine ovarian oocytes were fused with either homologous (porcine) or heterologous (rabbit) oocytes, both at different stages of maturation. The maturation-promoting factor (MPF) present in maturing porcine oocytes or ovulated rabbit oocytes induced rapid chromosome condensation of the oocytes with intact germinal vesicles (GVs). In the case of activation of ovulated rabbit oocyte, germinal vesicle breakdown (GVBD) of porcine oocytes was incomplete or did not occur. In the giant cells consisting of two immature porcine oocytes, meiotic maturation proceeded in the same manner as in unfused oocytes. However, in cells derived from fusion of immature porcine and rabbit oocytes, two metaphase groups of chromosomes were observed 6 h after fusion. It may be concluded that GVBD is governed after fusion by the cytoplasm originating from the oocytes of more advanced stages of maturation or from those which mature faster.     

ON THE ROLE OF CALCIUM IONS IN OOCYTE MATURATION IN THE POLYCHAETE CHAETOPTERUS PERGAMENTACEUS*

The germinal vesicle of mechanically released Chaetopterus oocytes disintegrates in natural sea water (NSW), but not in artificial sea water of normal composition (ASW), calcium-free sea water (CaFSW), magnesium-free sea water (MgFSW) or calcium and magnesium-free sea water (CaMgFSW). Several methods of inducing oocyte maturation using chemically well-defined medium have been established. (1) Germinal vesicle breakdown was induced by the treatment of immature oocytes with KCl (60 mM) in ASW or MgFSW. The presence of Ca²⁺ is necessary for inducing oocyte maturation with high potassium concentration. “Differentiation without cleavage” was observed after this treatment. (2) Trypsin (0.3%) induced oocyte maturation in ASW, but not in CaFSW. Oocytes matured in this manner developed to trochophores upon insemination. (3) Immature oocytes, treated with isotonic CaCl₂ for less than 1 min and then transferred to ASW, underwent germinal vesicle breakdown. The oocytes were arrested at the first meiotic metaphase and upon insemination developed to trochophore larvae. (4) Tetracaine (0.4 mM) induced oocyte maturation in the absence of Ca²⁺ in the medium. In ASW, CaFSW or CaMgFSW containing the drug, oocytes were arrested at the first meiotic metaphase, while in MgFSW with tetracaine they developed parthenogenetically up to the 4- and 8-cell stages. The role of calcium in oocyte maturation was established and its importance was discussed based on the results obtained with the different ways of inducing oocyte maturation.     

A correlation between juvenile hormone deficiency and vitellogenic oocyte degeneration inDrosophila melanogaster

Summary It is known from previous work that juvenile hormone (JH) is required to initiate vitellogenin uptake into maturing oocytes ofDrosophila melanogaster, but additional requirements for this hormone during oocyte maturation have not been fully understood. To determine if early vitellogenic oocytes (stages 8 and 9) require JH for continued development, these oocytes were transplanted toDrosophila female and male hosts which were rendered deficient in JH by three methods. Implanted stage 9 and usually stage 8 oocytes were found to degenerate in JH-deficient hosts unless ZR-515, a JH analogue, was applied to the host shortly after implantation.These results were confirmed during in situ ovary development. JH deficiency was produced in gravid females, and ovaries examined at subsequent time intervals were found to be deficient in stage 8–10 oocytes as early as 6 h after treatment. Degenerating oocytes corresponding to these stages were commonly found. ZR-515 prevented oocyte degeneration during at least the first 8 h and continued to support stage 8–10 oocyte development 24 h after application to these females. The results suggest that JH is required not only for initiation but also for continuation of vitellogenin uptake and oocyte development.     

显微受精废弃的人类卵母细胞体外成熟及孤雌激活

以卵胞浆单精注射(intracytoplasmic sperm injection,ICSI)后废弃的未成熟人类卵母细胞(生发泡期卵母细胞(the germinal vesicle,GV)和第一次减数分裂中期卵母细胞(the metaphase,MI))为材料,使用卵母细胞体外成熟培养液培养未成熟的卵母细胞,分别在人类绒毛膜促性腺激素(human chorionic gonadotrophin,hCG)注射后45、60、84 h观察卵母细胞成熟情况.分别使用钙离子载体(calcium ionophore,CI)A23187联合6-二甲基氨基嘌呤(6-DMAP)法或精子提取物卵胞质内注射(sperm extracts intracytoplasmic injection,SEII)法两种不同的激活方法对体外成熟MII的卵母细胞进行孤雌激活,评价其体外发育潜能.MI卵子体外成熟率要显著高于GV(75.2%vs 30.6%)(P<0.01).与CI/6-DMAP法相比使用SEII/6-DMAP法在激活率(87.5%vs 70.2%)上要明显高于CI/6-DMAP法(P<0.05),但在卵裂率(65.7%vs 72.5%)和桑囊率(0%vs 5.0%)上SEII/6-DMAP法要低于CI/6-DMAP法.注射hCG 45 h组的卵母细胞激活率(91.3%vs 57.9%)、卵裂率(85.7%vs 57.9%)及桑囊率(9.5%vs 0%)均显著高于注射hCG 60 h组(P<0.01).56.8%(117/206)的ICSI废弃的未成熟卵母细胞可以在体外发育成熟,激活后具有一定的发育潜能,卵龄对卵母细胞的质量和发育能力影响较大.     

Induction of superovulation in prepubertal female rats by anterior pituitary transplants

Transplants of 26-day-old rats of an anterior pituitary gland from adult intact or castrated male, 20-day-old or adult ovariectomized female donors (all of which contained large amounts of FSH) resulted in superovulation in recipients on the morning of Day 29. Transplants of the gland from 20-day-old males and adult cyclic females could not advance the time of first ovulation or induce superovulation. In the rats in which superovulation could be induced, a marked increase in plasma FSH was noted in recipients shortly after transplantation and the high levels of plasma FSH were maintained until at least 12 h after grafting. These rats also showed preovulatory surges of LH and FSH 54 h after grafting. No obvious elevation of plasma FSH was noted over 72 h in recipients in which superovulation could not be induced. These findings suggest that the final maturation of follicles for superovulation is induced by a transient release of a large amount of FSH from the grafted pituitary gland and that the sex of the pituitary donor has no bearing on this phenomenon.     

Effects of in vivo and in vitro exposure to bromocriptine on testicular LH receptors and in vitro testosterone production in Syrian hamsters.

The effects of in vivo and in vitro exposure to bromocriptine (CB-154) were studied in testes of Syrian hamsters. In animals treated for two days with CB-154, a decrease in LH receptors (LH-R) was observed, with a greater decrease being measured in hamsters treated for 14 days, when compared with controls. Injection of HCG caused, in hamsters treated with CB-154 for 14 days, up-regulation of LH-R and increased testosterone synthesis in response to HCG administration in vitro. These changes were not observed in the two other groups of animals. When testis fragments were incubated with CB-154, those incubated with a large dose (10 micrograms/ml) had a normal pattern of response to HCG, and those incubated with a small dose (1 ng/ml) had a smaller maximum response. These actions are similar to those observed in men treated with CB-154. It can be therefore concluded that: a) CB-154 has a direct effect on the testes; b) it probably is through modulation of LH-R synthesis; c) Syrian hamsters probably represent the best model for the study of the effects of CB-154 on the testes; and d) the possibility of using CB-154 as an adjuvant of gonadotropin treatment in hypogonadism has to be considered.     

The effect in vivo of alterations in gonadotropins and cyclic nucleotides on oocyte maturation in the hamster

The temporal relationship of changes in cAMP and cGMP to oocyte maturation was examined in proestrous hamsters (day 4). The first series of experiments showed, in normal cycling hamsters, an increase in cAMP and a decrease in cGMP at 1400 h shortly after the rise in LH with oocyte maturation beginning at 1800 h. When a second group of animals was injected with phenobarbital at 1200 h to block the LH surge, no significant change occurred in either cyclic nucleotide and oocyte maturation was prevented. In the second series of experiments single injections of either saline, hCG (30 IU), LH (10 micrograms) or FSH (10 micrograms) were given each to a group of animals at 0900 h on day 4. Animals were killed at five time intervals between 15 min and 3 h following the injection. LH and hCG stimulated a simultaneous increase in cAMP and decline in cGMP. The injection of FSH, however, did not cause an increase in cAMP but still produced a sharp decline in cGMP. Oocyte maturation occurred at 3 h in those animals injected with gonadotropins. Animals injected with saline showed neither cyclic nucleotide changes nor oocyte maturation. When cAMP and cGMP levels were expressed as a ratio (cAMP/cGMP) a significant increase occurred in the normal cycling animals and in those injected at 0900 h with gonadotropins. Phenobarbital and saline injected control animals showed no significant increase in the cAMP/cGMP ratio and no oocyte maturation. The results of these experiments and previous studies by this investigator indicate that cGMP may play an important role in oocyte maturation in the hamster prior to the LH surge. Since, in the presence of gonadotropins, the cAMP/cGMP ratio increases prior to oocyte maturation, it may be that the cyclic nucleotide ratio is also of importance in this process. Previous work by Hubbard and Terranova (1) has shown that guanosine 3':5' cyclic monophosphate (cGMP), can inhibit spontaneous maturation of hamster oocytes in vitro. This inhibitory action was dose dependent and overcome by LH. The cGMP-mediated inhibition occurred only in cumulus-enclosed oocytes, while adenosine 3':5' cyclic monophosphate (cAMP) inhibited spontaneous maturation in both cumulus-enclosed and denuded oocytes. The results of this study suggested that cGMP may play a role in inhibiting oocyte maturation prior to the LH surge. LH, the initiator of oocyte maturation, has also been shown in the intact proestrous rat and hamster to cause a decrease in cGMP at the same time that cAMP is rising (2,3).(ABSTRACT TRUNCATED AT 400 WORDS)     

Melatonin alleviates the deterioration of oocytes from mice subjected to repeated superovulation

Induction of repeated superovulation with exogenous hormones is widely used in assisted reproductive technology (ART). Though it is generally safe, emerging evidence has indicated that repeated superovulation may compromise oocyte quality. However, few studies have explored how to ameliorate such impairment. Because melatonin has beneficial influences on oocytes in various detrimental environments, we aimed to explore whether melatonin could protect mouse oocytes after repeated superovulation. We found that repeated superovulation markedly reduced meiotic maturation and disrupted spindle organization and chromosome alignment. Furthermore, we observed reduced mitochondrial content and enhanced early apoptosis in oocytes from mice subjected to repeated superovulation. In addition, 5-methylcytosine (5mc) fluorescence intensity was lower in oocytes from experimental mice than in those from control mice, indicating that repeated superovulation disrupts genomic DNA methylation, and elevations in reactive oxygen species levels indicated that repeated superovulation also induces oxidative stress. Conversely, melatonin administration improved oocyte maturation and attenuated the observed defects. Interestingly, supplementation with melatonin during in vitro maturation had the same protective effects on oocytes as in vivo melatonin administration. In summary, our results show that melatonin can improve oocyte quality after repeated superovulation and thus provide a potential strategy to improve ART efficiency.