Compensatory development in preimplantation mouse embryos derived from delayed mating.

Rodent embryos resulting from delayed mating grow relatively faster than those resulting from normal mating. To evaluate this phenomenon quantitatively, in the present study we compared the number of cells at the preimplantation stage of mouse embryos derived from normal mating and those derived from delayed mating (3 and 6 h after ovulation). The mean cell numbers (45.4 and 43.0 for delayed mating at 3 and 6 h postovulation, respectively) of embryos at 77 h postcoitus (hpc) in the delayed mating groups were greater than that (38.4) of the normal mating group. Further, when the mean cell numbers (38.8 and 38.5) in the delayed mating groups were counted at 74 hpc, they were almost equal to that of the normal mating group at 77 hpc. The study demonstrated that preimplantation mouse embryos derived from delayed mating progress more rapidly than their normally mated counterparts. However, a 3-h advance in development seems to be the limit of this increased rate of growth, even when the time interval from ovulation to mating is longer than 3 h. The mechanism(s) of this interesting compensatory phenomenon should be investigated.     

Increased mortality during early embryonic development after in-vitro fertilization of rat oocytes

Immature female rats (60-65 g) were injected with 4 i.u. PMSG on Day -2, and allocated to 3 groups. For Groups I and II, unmated donors were killed 67-69 h after PMSG injection, shortly after the expected time of ovulation. Oocytes were recovered from the oviducts and transferred immediately into the oviduct of mated recipients (Group I) whose ipsilateral ovary had been exposed by peeling back the bursa, preventing endogenous oocytes from entering the oviduct, or were fertilized in vitro (Group II) and were transferred 16-18 h later. Rats in Group III were allowed to mate and half were killed 6 h after mating. The fertilized oocytes were then incubated for 10-12 h until transfer. The remaining rats in Group III were killed 16-18 h after mating and fertilized oocytes were collected and transferred immediately. Recipient rats were killed on Days 2, 5, 8 and 20. Zygotes resulting from in-vitro fertilization (Group II) were as able as those fertilized in donors (Group III) or recipients (Group I) to develop to the 2-cell stage, but underwent significantly greater embryonic loss beyond this stage of development. There was a slower rate of development of such oocytes to the blastocyst stage (Day 5) and a lower mean weight of implantation sites (Day 8). Transfer of zygotes after in-vitro fertilization resulted in a loss of 35% of the embryos at the time of implantation. These results suggest that in-vitro fertilization of rat oocytes leads to defects in the embryos causing a delay in early embryo development and a large number of implantation losses.     

Effects of immunization against bone morphogenetic protein 15 and growth differentiation factor 9 on ovulation rate, fertilization, and pregnancy in ewes

Immunization of ewes against growth differentiation factor 9 (GDF9) or bone morphogenetic protein 15 (BMP15) can lead to an increased ovulation rate; however, it is not known whether normal pregnancies occur following such treatments. The aims of the present study were to determine the effects of a short-term immunization regimen against BMP15 and GDF9 on ovulation rate, fertilization of released oocytes, the ability of fertilized oocytes to undergo normal fetal development, and the ability of immunized ewes to carry a pregnancy to term. Ewes were given a primary and booster immunization against keyhole limpet hemocyanin (KLH; control, n = 50), a GDF9-specific peptide conjugated to KLH (GDF9, n = 30), or a BMP15-specific peptide conjugated to KLH (BMP15, n = 30). The estrous cycles of all ewes were synchronized, and ewes were joined with fertile rams approximately 14 days after the booster immunization. The number of corpora lutea was determined by laparoscopy 3-4 days following mating. Subsequently, about one-half of the ewes in each group underwent an embryo transfer procedure 4-6 days following mating, with the embryos being transferred to synchronized, nonimmunized recipients. The remaining ewes were allowed to carry their pregnancies to term. Short-term immunization against either BMP15 or GDF9 peptides resulted in an increase in ovulation rate with no apparent detrimental affects on fertilization of released oocytes, the ability of fertilized oocytes to undergo normal fetal development, or the ability of the immunized ewes to carry a pregnancy to term. Therefore, regulation of BMP15, GDF9, or both is potentially a new technique to enhance fecundity in some mammals.     

First cell cycle of zygotes of the mouse derived from oocytes aged postovulation in vivo and fertilized in vivo

This paper describes an analysis of the first cell cycle of mouse oocytes aged postovulation and fertilized in vivo. For this purpose, we developed a procedure for inducing ovulation in vivo that allows accurate timing of ovulation. The method is based on a luteinizing hormone (LH)-releasing hormone (LHRH) administration at proestrus. This ovulation procedure had no detectable effect on the rate of ovulation or postimplantation embryonic death. We used this method of ovulation induction in an analysis of the separate stages of the first cell cycle of in vivo fertilized postovulation aged oocytes. All stages assessed were shorter in aged oocytes (12 hr postovulation) than in zygotes from unaged oocytes (1 hr postovulation): 1) the time interval between insemination and penetration of the aged oocytes was 1.5 hr shorter than the time interval of the unaged oocytes; 2) pronuclear formation in the fertilized aged oocytes was somewhat quicker than pronuclear formation in fertilized unaged oocytes; 3) in zygotes from aged oocytes, the time between formation of pronuclei and the pronuclear membrane breakdown was 1 hr shorter than in zygotes from unaged oocytes; 4) the first cleavage division was 3 hr advanced in zygotes from aged oocytes compared with the moment of the first cleavage division in zygotes from unaged oocytes. We also determined the glutathione (GSH) content of unaged and aged oocytes to investigate a possible relationship between the rate of pronuclear formation and GSH. The level of GSH was two times lower in oocytes aged postovulation for 12 hr than in unaged oocytes.2+ level of GSH in fertilized, unaged oocytes was half that in     

Fertilization rates in superovulated and spontaneously ovulating mares

Embryo recovery per ovulation has been shown to be lower in superovulated mares than in untreated controls. The objectives of this study were to 1) determine whether follicles stimulated with superovulatory treatment ovulate or luteinize without ovulation, 2) determine fertilization rates of oocytes in oviducts of superovulated and control mares, and 3) evaluate viability of early stage embryos from superovulated and control mares when cultured in equine oviductal cell-conditioned medium. Cyclic mares were randomly assigned to 1 of 2 groups (n=14 per group) on the day of ovulation (Day 0): Group 1 received 40 mg of equine pituitary extract (EPE; i.m.) daily beginning on Day 5 after ovulation; mares assigned to Group 2 served as untreated controls. All mares were given 10 mg PGF(2alpha) on Day 5 and Day 6, and 3,300 IU of human chorionic gonadotropin (hCG) were administered intravenously once mares developed 2 follicles >/=35 mm in diameter (Group 1) or 1 follicle >/=35 mm in diameter (Group 2). Mares in estrus were inseminated daily with 1 x 10(9) progressively motile spermatozoa once a >/=35 mm follicle was obtained. Two days after the last ovulation the ovaries and oviducts were removed. Ovaries were examined for ovulatory tracts to confirm ovulation, while the oviducts were trimmed and flushed with Dulbeccos PBS + 10% FCS to recover fertilized oocytes. All fertilized oocytes (embryos) recovered were cultured in vitro for 5 d using TCM-199 conditioned with equine oviductal cells. Ninety-two percent of the CL's from EPE mares resulted from ovulations compared with 94% for mares in the control group (P>0.05). The percentages of ovulations resulting in embryos were 57.1 and 62.5% for EPE-treated and control mares, respectively (P>0.05). Eighty-eight (Group 1) and 91% (Group 2) of the freshly ovulated oocytes recovered were fertilized (P>0.05). After 5 d of culture, 46.4 and 40.0% of the embryos from EPE-treated and control mares developed to the morula or early blastocyst stage (P>0.05). In summary, the CL's formed in superovulated mares were from ovulations not luteinizations. Although embryo recovery was less than expected, fertilization rates and embryo development were similar (P>0.05) between superovulated and control mares.     

Status of oocytes and embryos of X-autosome translocation carrier sows

The impact of an X-autosome translocation t(Xp+; 14q-), on ovulation, fertilization and embryo survival in carrier sows, was examined and compared with these parameters of normal sows. Corpora lutea counts during week-2 and week-4 of gestation were similar in normal and carrier sows (14.4 +/- 1.36 and 15.5 +/- 2.18) although embryo recovery (11.0 +/- 1.87 and 6.0 +/- 1.47) was lower than that from normal sows (12.8 +/- 1.46 and 11.5 +/- 0.87), at these stages. Among the embryos karyotyped from the week-2 embryos of carrier sows, 42% were normal, 26.4% were carriers and 31.6% were of unbalanced chromosome make-up, and of the week-4 embryos of carriers, 33.3% were normal, 57.1% were carriers and 9.1% were chromosomally unbalanced females. The preponderance of females among the unbalanced embryos recovered at week-2 of gestation (11_ and 1_) and the total absence of males among those recovered at week-4, suggest that oocytes with unbalanced chromosome constitution are eliminated before week-2 of gestation if they are fertilized by Y bearing sperm, and that the unbalanced oocytes fertilized by X bearing sperm survive up to the peri-attachment stage even though all chromosomally unbalanced embryos are eliminated before term regardless of their sex.     

Development of embryos from in vitro ovulated and fertilized oocytes of the quail (Coturnix coturnix japonica)

The development of quail embryos obtained after in vitro fertilization of oocytes ovulated in vitro was investigated. About 40% of the specimens, after 18-20 hr of incubation, had undergone cleavage to reach stages IV-VI when viewed under a stereo microscope. However, only 36% of these embryos contained normal, DAPI-stained nuclei when observed under a fluorescent microscope; the other 64% showing a morphologically normal cleavage pattern did not contain nuclei. Control unfertilized oocytes, ovulated in vitro and cultured for the same time, also sometimes attained the morphologically correct stages IV-VI but their "blastomeres" were always devoid of nuclei. Therefore, it is advisable to monitor early avian embryos for the presence of nuclei when assessing development in culture.The results demonstrate, for the first time, that cytoplasmic segmentation can occur in the absence of nuclear divisions in the germinal disc of the quail and show the existence and significance of ooplasmic maternal information in birds. This phenomenon is also known for sea urchin and frogs. It is indicative of the role of maternal information in early development. The in vitro method presented here links the steps of ovulation and fertilization with the early cleavage stages under in vitro conditions and may be useful in studying mechanisms of fertilization and differentiation in birds as well as in obtaining transgenic birds by DNA injection or application of foreign, DNA-carrying sperm.     

Ovulation and fertilization of primary and secondary oocytes in LT/Sv strain mice

In this study, the chromosome constitution of both unfertilized oocytes and fertilized eggs isolated from the oviducts of LT/Sv strain mice were analyzed. Air-dried chromosome preparations from unfertilized oocytes revealed that about one-third of those examined were ovulated as primary oocytes. These were arrested at metaphase of the first meiotic division and exhibited the characteristic “tetrad” chromosome configuration. The remaining two-thirds of the unfertilized oocytes were ovulated at metaphase of the second meiotic division. The fertilized eggs were isolated from the oviducts of LT/Sv females previously mated to (C57BL × CBA) F1 hybrid males. Analysis of the fertilized eggs at metaphase of their first cleavage mitosis revealed that about one-third of the eggs examined were digynic triploids, whereas the remaining two-thirds had the normal diploid chromsome constitution. In the triploids, the 40 female chromosomes present (mouse, n = 20) were derived from a single diploid pronucleus formed after the extrusion of a first polar body, and following the monospermic fertilization of primary oocytes. The female pronuclear-derived chromosomes invariably exhibited “homologous pairing,” and these were associated at their centromeres. The ovulation, penetration, and subsequent fertilization of primary oocytes is an extremely unusual phenomenon in mammals and only appears to occur on a regular basis in LT/Sv mice. The premature “cytoplasmic maturation” of these oocytes is of interest, as they clearly have the same developmental capacity as secondary oocytes. The significance of these observations in relation to folliculogenesis and litter size in LT/Sv mice is discussed.     

Fertilization and development of quail oocytes after intracytoplasmic sperm injection

The present study was conducted to establish the intracytoplasmic sperm injection (ICSI) method for in vitro fertilization and development in quail. The efficiency of fertilization of oocytes was compared 1) between spontaneous and premature ovulation and 2) among testicular round spermatids, elongated spermatids, and immature and mature spermatozoa. The oocytes were injected with a single spermatozoon or spermatid and cultured for 24 h. Cell division was histologically observed with hematoxylin-eosin (HE) and a nucleus-specific fluorescent dye (DAPI). Five of 30 (16.6%) and 4 of 30 (13.3%) oocytes injected with mature sperm were fertilized in the spontaneous and induced ovulation group, respectively. Those embryos showed development at stages II-VII. Half the number (three of six) of the oocytes injected with testicular spermatozoa were fertilized and developed to stages IV-VII, and two of five oocytes injected with elongated spermatids were fertilized and developed to stage VI. All ooocytes injected with round spermatids were unfertilized. The results demonstrate that intracytoplasmic injection of a single sperm into quail oocyte can activate the oocyte and lead to fertilization. Oocytes prematurely ovulated are capable of fertilizing with mature sperm as are those spontaneously ovulated. In addition, the results suggest that the testicular round spermatids may not possess sufficient oocyte-activating potency but that the elongated spermatids and immature spermatozoa are competent to participate in fertilization and early embryonic development in quail.     

第二极体排出时间早晚与胚胎发育潜能相关性的研究

目的探讨第二极体排出时间早晚与胚胎质量及发育潜能之间的关系。方法以本生殖医学中心2009年6月-8月IVF—ET周期患者受精卵子为研究对象,共计1170枚卵子。以受精5h为时间界限将受精胚胎分为第二极体正常排出组（正常组）和延迟排出组（延迟组）。分别观察两组卵子正常／异常受精率（2PN率,1PN和3PN率）和优质胚胎率;同时统计阳性妊娠结局所移植胚胎中,正常组和延迟组的胚胎比例各占多少。采用卡方检验对数据进行统计学处理。结果①两组正常受精卵数目之间以及异常受精卵数目之间均有显著性差异（P〈0．05）;两组总受精卵数目之间有非常显著差异（P〈0．001）。②两组的优质胚胎率之间显著性差异（P〈0．05）。③统计阳性妊娠结局所移植的97个胚胎中,来自于正常组的胚胎有92个（94．9％）,仅5个是来自于延迟组（5．1％）。结论受精5h内排出第二极体的卵子其总受精率、正常受精率以及所发育的胚胎质量均显著高于第二极体出现晚的卵子,而且有着较高的胚胎植入率。对受精5h的卵子进行第二极体观察有助于早期预测患者本次IVF-ET周期胚胎的发育潜能以及妊娠结局;还可以作为决定是否行早补救ICSI的判定指标之一。     

Effects of delayed mating on preimplantation embryos in spontaneously ovulated mice

The effects of delayed mating on mouse preimplantation embryos (78 ± 1 hours) were studied by setting up different mating periods in relation to the estimated time of spontaneous ovulation. Copulation occurred even in the late morning and early afternoon after the night of spontaneous ovulation. However, females mated in the early afternoon had no viable embryos at the time of laparotomy. Although embryonic development was not affected in the groups mated 6 or 10 hours after estimated ovulation, the percentage of degenerated embryos was increased in these groups. These results suggest that prolonged intervals between the estimated time of ovulation and mating have some deleterious effects on preimplantation embryos.     

Molecular differentiation of the rabbit ovum. III. Fertilization-autonomous polypeptide synthesis

Autoradiographic patterns of [1-³⁵S]methionine-labeled polypeptides separated by two-dimensional polyacrylamide gel electrophoresis were obtained from (1) rabbit oocytes that had undergone meiotic maturation to metaphase II in vivo or in vitro, (2) in vitro matured oocytes cultured for an additional 36 hr, or recovered from the reproductive tract at 36 hr after ovulation, (3) newly fertilized eggs, and (4) embryos developed in vivo or in vitro from the 1-cell stage to the 12- to 16-cell stage. The findings indicate that the detectable synthesis of a set of stage-specific (cleavage) polypeptides is autonomous of fertilization and appears to follow a timed, translational schedule initiated with the breakdown of the oocyte nucleus during the resumption of arrested meiosis.     

The influence of postovulatory age on the rate of cleavage in in-vitro fertilized rat oocytes

The purpose of this study was to analyze the effect of postovulatory ‘aging’ in the oviduct on the rate of zygotic development. Two ovulatory ages were tested: oocytes collected from the oviducal ampullae 1) soon after ovulation (denoted freshly ovulated) or 2) 7-hour postovulation. All the oocytes were from superovulated immature rats. By manipulation of the timing of the ovulatory hormone treatment, it was possible to place both types of oocytes into sperm suspension from the same pool and at the same time. The oocytes and spermatozoa were coincubated overnight. Cleavage was established by interference contrast microscopy. The time of the first cleavage of ova from the 7-hour postovulation group was clearly advanced. Because the cleavage time curves were not parallel, no reliable estimate of the time difference could be made, but it was clearly in the range of 2 hr. This shift could not be related to any difference in the time of sperm penetration. Both groups of oocytes underwent penetration by spermatozoa at the same time. The time interval between maximal sperm penetration (94% of oocytes in both groups) and maximal cleavage (50% in both groups) was 23 hr in the freshly ovulated and 21 hr in the 7-hour postovulatory eggs. Nor was the difference related to polyspermy, which was approximately 14% in both groups. These results support the hypothesis that developmental processes are under way in the oocyte before fertilization, but at a much slower rate than after fertilization.     

Embryonic loss in superovulated cattle caused by the 1;29 Robertsonian translocation

The effect of the 1;29 Robertsonian translocation on fertility was studied using embryos resulting from matings of nine carrier cows and two carrier bulls. Embryos were collected from the following three mating groups utilizing superovulation: normal bull cross normal cow, normal bull cross translocation carrier cow, and translocation carrier bull cross normal cow. The proportion of ova which were fertilized did not vary among the groups, indicating that fertilization rates were not affected by the translocation. The translocation cows did yield fewer embryos on average than did cows with normal karyotypes, which may suggest ovulation rates are reduced (at least after superovulation attempts) in cattle carrying the 1;29 translocation. Twenty of 39 embryos successfully karyotyped had abnormal chromosome complements. All four of the theoretically predicted karyotypes and two additional abnormal combinations were found. Eight of 39 (20.5%) embryos karyotyped had unbalanced karyotypes which would have resulted in embryonic loss. The proportion of embryos with unbalanced karyotypes, was slightly higher when the cow (36%) carried the translocation than when the bull (19%) did. Results of this study indicate that fertility is impaired due to the presence of this translocation. The major loss in reproductive potential appears to be due to embryonic loss rather than fertilization failure.     

Successful pregnancies after transfer of embryos recovered from ewes induced to ovulate 24-29 days post partum

After lambing in late November, oestrus and ovulation were induced by using a CIDR device and PMSG in early weaned (N = 13) or lactating (N = 14) Border Leicester x Scottish Blackface ewes between 23 and 29 days after parturition. Ewes were intrauterine inseminated under laparoscopic visualization 54-55 h after CIDR-device withdrawal and eggs recovered on Day 3 of the cycle. Ovum recovery and fertilization rates were higher in lactating than in early weaned ewes, with fertilization being achieved as early as 24 days post partum in both groups. Of the 7 early weaned and 11 lactating ewes yielding eggs, fertilization occurred in 4 and 7 ewes respectively. A total of 20 embryos were transferred to the normal uterine environment of 15 recipient ewes in which the interval from parturition was greater than 150 days. Pregnancies were successfully established in 9 recipient ewes, resulting in the birth of 10 viable lambs. Prolactin concentrations were significantly higher (P less than 0.001) in lactating than in early weaned ewes throughout the study. Nevertheless, normal luteal function (as assessed by daily progesterone concentrations) was exhibited by 12 of 14 lactating and 8 of 13 early weaned ewes. Two post-partum donors in which the corpora lutea completely failed to secrete progesterone yielded fertilized eggs which developed to term when transferred to a normal uterine environment. The results show that sheep oocytes can be fertilized using laparoscopic intrauterine insemination as early as 24 days after parturition and that the resulting embryos are viable when recovered on Day 3 after oestrus and transferred to a normal uterine environment.     

Effect of time of ovulation and sperm concentration on fertilization rate in gilts

In normal production practices, sows and gilts are inseminated at least twice during estrus because the timing of ovulation is variable relative to the onset of estrus. The objective of this study was to determine if a normal fertilization rate could be achieved with a single insemination of low sperm number given at a precise interval relative to ovulation. Gilts (n=59) were randomly assigned to one of three treatment groups: low dose (LD; one insemination, 0.5 x 10(9) spermatozoa), high dose (HD; one insemination, 3 x 10(9) spermatozoa) or multiple dose (MD; two inseminations, 3 x 10(9) spermatozoa per insemination). Twice daily estrus detection (06:00 and 18:00 h) was performed using fenceline boar contact and backpressure testing. Transrectal ultrasonography was performed every 6 h beginning at the detection of the onset of standing estrus and continuing until ovulation. Gilts in the LD and HD groups were inseminated 22 h after detection of estrus; MD gilts received inseminations at 10 and 22 h after detection of estrus. Inseminations were administered by using an insemination catheter and semen was deposited into the cervix. The uterus was flushed on Day 5 after the onset of estrus and the number of corpora lutea, oocytes, and embryos were counted. Time of insemination relative to ovulation was designated as 40 to >24 h, 24 to >12 h, and 12 to 0 h before ovulation and >0 h after ovulation. The LD gilts had fewer embryos (P<0.04), more unfertilized oocytes (P<0.05) and a lower fertilization rate (P<0.07) compared to MD gilts. The effects of time of insemination relative to ovulation and the treatment by time interaction were not significant. We conclude that a cervical insemination with low spermatozoa concentration may not result in acceptable fertility even when precisely timed relative to ovulation.     

An essential role for functional telomeres in mouse germ cells during fertilization and early development

Late generations of telomerase-null (TR(-/-)) mice exhibit progressive defects in highly proliferative tissues and organs and decreased fertility, ultimately leading to sterility. To determine effects of telomerase deficiency on germ cells, we investigated the cleavage and preimplantation development of embryos derived from both in vivo and in vitro fertilization of TR(-/-) or wild-type (TR(+/+)) sperm with either TR(-/-) or TR(+/+) oocytes. Consistently, fertilization of TR(-/-) oocytes with either TR(+/+) or TR(-/-) sperm, and TR(-/-) sperm with TR(+/+) oocytes, resulted in aberrant cleavage and development, in contrast to the normal cleavage and development of TR(+/+) oocytes fertilized by TR(+/+) sperm. Many (>50%) of the fertilized TR(-/-) eggs developed only one pronucleus, coincident with increased incidence of cytofragmentation, in contrast to the normal formation of two pronuclei and equal cleavage of wild-type embryos. These results suggest that both TR(-/-) sperm and oocytes contribute to defective fertilization and cleavage. We further found that a subset (7-9%) of telomeres was undetectable at the ends of some metaphase I chromosomes from TR(-/-) spermatocytes and oocytes, indicating that meiotic germ cells lacking telomerase ultimately resulted in telomere shortening and loss. Dysfunction of meiotic telomeres may contribute to aberrant fertilization of gametes and lead to abnormal cleavage of embryos, implying an important role of functional telomeres for germ cells undergoing fertilization and early cleavage development.     

The effects of spermatozoal irradiation with X-rays on chromosome abnormalities and on development of mouse zygotes after delayed fertilization

The chromosome complements of zygotes derived from oocytes aged post ovulation and fertilized in vivo with X-ray-irradiated sperm were studied. Ovulation was induced by an injection of luteinizing hormone-releasing hormone (LHRH) at pro-estrus and fertilization was achieved by artificial insemination at 13 h and 24 h after LHRH in order to obtain embryos from unaged and aged (12 h post-ovulation) oocytes respectively. Post-ovulatory aging prior to fertilization did not significantly affect the percentage of zygotes with irradiation-induced chromosome abnormalities. However, post-ovulatory aging had a negative effect on the morphology of male as well as female pronuclear chromosomes of the first cleavage metaphase. When fertilized with control spermatozoa this effect was apparent in both the male and the female pronucleus. When unaged oocytes were fertilized with X-irradiated spermatozoa chromosome morphology was also adversely affected in both pronuclei. In zygotes from aged oocytes, there was an extra negative effect of X-rays on the male pronuclear chromosomes only. After fertilization with X-irradiated sperm 27% of zygotes from aged oocytes were arrested at interphase compared to 7% from unaged oocytes. We suggest that post-ovulatory aging and X-rays affect the male and female pronuclear chromatin structure after fertilization. These chromatin alterations could interact with DNA lesions induced in the spermatozoa prior to fertilization, such that development to first cleavage can be blocked.     

A study of factors affecting the success of human fertilization in vitro. II. Influence of semen quality and oocyte maturity on fertilization and cleavage

The incidence of in vitro fertilization was analyzed with respect to the degree of cumulus dissociation (expansion) at the time of oocyte recovery and also the semen quality. Of the oocytes surrounded by perfectly ("++") or moderately ("+") dissociated cumuli, 78.6% and 30.8%, respectively (P less than 0.001), were fertilized when the husband's semen analysis was in the normal range. The proportion of fertilized oocytes was not decreased in cases of polyzoospermia (greater than 130 X 10(6) spermatozoa/ml), but was decreased (P less than 0.05) when the semen analysis revealed other anomalies: oligozoospermia (less than 15 X 10(6) spermatozoa/ml), asthenozoospermia (less than 50% motile cells) or teratozoospermia (greater than 50% abnormal spermatozoa). The proportion of fertilized eggs cleaving in vitro was unaffected by semen quality but was lower when "+" cumulus oocytes were collected than when "++" cumulus oocytes were obtained (58.3% vs. 87.0%, P less than 0.02). In vitro incubation of the oocyte prior to insemination increased the incidence of fertilization by about 28% for both "+" (22.2 to 50.0%) and "++" (65.7 to 93.9%) cumulus oocytes. Finally, 67.6% of "++" cumulus oocytes developed into embryos when the insemination with spermatozoa from normal semen samples was delayed by several hours, compared with only 29.0% when the conditions were suboptimal ("+" cumulus oocyte, abnormal semen analysis or no delay prior to insemination). Eight pregnancies began following the replacement of 38 embryos in 34 patients. Six spontaneous abortions occurred, and chromosomal abnormalities were proven in the two cases analyzed. Two pregnancies continued for more than 3 months, resulting in term deliveries of two normal babies.     

Reversal of postmortem degeneration of mouse oocytes during meiotic maturation in vitro

The developmental capacity of oocytes matured in vitro following isolation at the germinal vesicle stage from freshly killed mice (control) was compared with that of oocytes isolated from the carcasses of mice killed 3, 6, 9, and 12 hr earlier. The yield of intact, cumulus cell-enclosed oocytes decreased as the interval between death of the animal and removal of the ovary increased. After 15-16 hr of culture of medium containing follicle-stimulating hormone, the frequency of germinal vesicle breakdown, extrusion of a polar body, and cumulus expansion was equivalent in oocytes of all groups. The frequency of development of inseminated ova to 2-cell stage embryos in the control, 3, and 6 hr postmortem groups was the same but declined markedly in the 9 and 12 hr groups. There was also no difference in the frequency of blastocyst development from 2-cell stage embryos between the control, 3, 6, and 9 hr postmortem groups, but the 2-cell embryos in the 12 hr postmortem group did not develop to blastocysts. Thirty-six percent of the 2-cell stage embryos from the 6 hr postmortem group developed to live young after transfer to foster mothers. Follicles of 6 hr postmortem ovaries showed degeneration manifested as prominent crystalline inclusions within the oocytes and many pyknotic granulosa cells. The crystals disappeared within 1 hr of culture and the secondary oocytes appeared normal. The cultured oocyte-cumulus cell complexes, therefore, reversed degenerative changes induced by the death of the animal. This study demonstrates the feasibility of recovering developmentally competent oocytes from valuable recently deceased zoological, agricultural, and endangered mammals.