Developmental ability of porcine ova matured in porcine follicular fluid in vitro and fertilized in vitro

Developmental ability of porcine ova matured in porcine follicular fluid (pFF) with FSH in vitro and fertilized in vitro was examined by culturing in BMOC-2. Forty-eight hours after insemination, 35.6% of ova cleaved normally, and this rate was significantly higher (13.0%) than that of the ova matured in a modified Krebs-Ringer bicarbonate solution. Twenty-four percent (29 120 ) of ova matured in pFF with FSH developed to the four-cell stage and two of them developed to the eight-cell stage 66 h after insemination. Most cleaved embryos stopped developing at the four-cell stage and neither the morula nor blastocyst stage was observed throughout the culture period as reported in the in vivo matured ova. In culture at 37 degrees C, the appearance of two-cell and four-cell embryos was delayed from that of in vivo embryos, but their development was significantly accelerated by culturing at 39 degrees C. These results show that pFF is an excellent maturation medium for porcine oocytes, and the developmental capacity of the ova matured in pFF seems to be similar to that of in vivo matured ova. Culturing at 39 degrees C was found to be more suit-able for the development of ova than 37 degrees C.     

Effect of maternal age and parity on preimplantation embryo development and transport in the golden hamster (Mesocricetus auratus)

Preimplantation embryo development was studied in the golden hamster (Mesocricetus auratus). Three groups of regularly cycling female hamsters were used: (I) 30 nulliparous young female (NYF) hamsters; (II) 24 nulliparous adult female (NAF) hamsters and (III) 30 multiparous adult female (MAF) hamsters. Female hamsters were mated with male hamsters of proven fertility. Only 15 min were allowed for mating. The moment of ejaculation was registered. Female hamsters were killed from 60 to 69 h after coitus. Corpora lutea were counted in both ovarian surfaces. Oviducts and uterine horns were flushed separately and embryo number, stage of development and distribution were recorded. Adult female hamsters, nulliparous and multiparous, had significant higher ovulation rates than NYF, but their reproductive efficiency was significantly lower. Preimplantation embryo development and transport were highly synchronous in NYF, but not in adults. Morulae were observed in NYF as early as 62-63 h after coitus. In adult female hamsters, significant numbers of morulae were found until 66-67 h. On the contrary, in NYF four-cell embryos were detected only until 60-61 h, while four-cell embryos were found until 64-65 h in NAF, and until 66-67 h in MAF. Embryo transport from the oviduct to the uterus is practically completed at 62-63 h after coitus in NYF, while it is evidently retarded in adult animals. In NYF all eight-cell embryos reached the uterus by 62 h after coitus. In adult female hamsters, both nulliparous and multiparous, a considerable number of eight-cell embryos fail to migrate into the uterus even at 67 h after coitus.     

A detailed analysis of early events during in-vitro fertilization of bovine follicular oocytes

Bovine follicular oocytes collected at slaughter were matured and fertilized in vitro with in-vitro capacitated spermatozoa. Analysis of 621 penetrated ova fixed at various times after in-vitro insemination led to definition of 6 stages of early development. A time sequence for sperm penetration, sperm head decondensation, male pronucleus formation, the activation of second meiotic division, female chromosome decondensation and pronucleus development was established. First sperm penetration into the ooplasm was recorded 6 h after insemination; 1-2 h was required for the sperm head to decondense and another 4-6 h to develop into the opposing pronucleus stage. Synkaryosis and first cleavage occurred 28 h after fertilization. Examination of the early stages revealed four types of abnormalities, i.e. polyspermy, polygyny, asynchrony between male and female pronucleus development, and preactivation of cytokinesis.     

Adrenalectomy: Effects on mating behavior,ovulation, and early gestation in the cycling rat

Adrenalectomy was performed 4 weeks prior to evaluating the effects on mating behavior, ovulation, and the first 10 days of gestation in 4-day cycling female rats. Mating behavior was essentially normal and occurred at times similar to sham-adrenalectomized animals on the afternoon of predicted proestrus. The number of ova ovulated was significantly reduced in mated adrenalectomized subjects in contrast to data reported by Rodgers (1971) for intact females mated at similar times in proestrus. Embryonic swellings were significantly fewer at day 10 of gestation than in the sham-adrenalectomized group providing additional evidence of reduced numbers of ova ovulated. Embryonic weights at day 10 of gestation were not affected by adrenalectomy. Removal of adrenocortical hormones significantly increased uterine weight on the morning of vaginal estrus. The data fail to show a significant delay in sexual receptivity as a result of removing adrenal progesterone, but suggest that adrenalectomy interferes with the neuroendocrine reflex mediating ovulation.     

Ovulation, fertilization and lambing rates, and peripheral progesterone concentrations, in ewes inseminated at a natural oestrus during November or February.

The objective of this study was to determine the relative importance of seasonal changes in ovulation rate, fertilization rate and embryo survival as the cause of reduced lambing rates in ewes mated in February compared with those mated in November. The study was conducted at 57 degrees N using mature Mule ewes and Suffolk rams. Sixty ewes were allocated equally to five groups: unbred (UB) or mated at a natural oestrus during November (N) or February (F) by natural (N) or cervical artificial (A) insemination. Groups were maintained separately at pasture supplemented with hay. A raddled vasectomized or non-vasectomized ram was present with UB, NN and NA groups from 26 October 1995 to 1 January 1996 and with UB, FN and FA groups from 25 January 1996 to 31 March 1996. Ewes marked by the ram were recorded twice a day, and those in groups NN, NA, FN and FA were inseminated at their second behavioural oestrus. For all ewes, blood samples were obtained once a day from introduction of the vasectomized rams until 30 days after mating (groups NN, NA, FN and FA) or 20 days after the first oestrus (group UB), and ovulation rate was measured by laparoscopy 7 days after the first oestrus. For ewes in groups NN, NA, FN and FA, ovulation rate was measured again after the second oestrus and ova were recovered from six ewes per group for assessment of fertilization before autotransfer. Pregnancy and lambing rates were recorded at term. Mean (+/- SE) dates of the first recorded oestrus for ewes in groups NN, NA and UB, and FN, FA and UB were 4 +/- 1.1 November and 4 +/- 0.9 February, respectively, and intervals between the first and second oestrus were 16 +/- 0.2 and 17 +/- 0.3 days (P < 0.01), respectively. Ovulation rates were 2.6 +/- 0.08 and 2.0 +/- 0.05 (P < 0.001), and peripheral progesterone concentrations during the luteal phase were 8.5 +/- 0.25 and 7.6 +/- 0.31 ng ml-1 (P < 0.05), for November and February, respectively. The difference in peripheral progesterone concentration was not solely attributable to the difference in ovulation rate. There was no significant effect of month or method of insemination, or of embryo recovery and autotransfer procedures on pregnancy rates and the proportion of ewes that became pregnant were NN 0.92, NA 0.83, FN 0.67 and FA 0.75. For ewes undergoing embryo recovery and autotransfer, ova recovered per corpus luteum were 1.00, 0.93, 1.00 and 0.92, fertilized ova per ovum recovered were 0.69, 0.92, 1.00 and 0.83, and lambs born per corpus luteum were 0.62, 0.79, 0.78 and 0.58 for NN, NA, FN and FA groups, respectively. There were no significant seasonal effects on fertilization rate or embryo survival. It is concluded that a seasonal decline in ovulation rate is the primary cause of reduced lambing rates in ewes mated in February compared with those mated in November. Pregnancy rates were high after mating in both periods and were not enhanced by the use of cervical insemination.     

A note on the reproductive success of primiparous blue fox vixens in social groups

Our aim was to compare traditional breeding system, i.e. artificial insemination with singly-housing, to alternative breeding systems in farmed blue fox (Alopex lagopus or Vulpes lagopus) vixens. At the age of 7 weeks (i.e. at weaning), 48 randomly selected female blue fox cubs were divided into four experimental groups: (1) artificially inseminated singly-housed vixens in cages (AI-SC), (2) artificially inseminated pair-housed vixens in double-cages (AI-PC), (3) naturally bred pair-housed vixens with a male in triple-cages (NB-PC), and (4) naturally bred pair-housed vixens with a male in outdoor enclosures (NB-PE). The cubs were counted on days 1, 2, 3, 7, 14 and 49 postpartum and the reproductive performance per breeding (RPB) and per mated vixens (RPM) with its subcomponents were formed from these data. RPB was zero in both NB groups. In AI-PC and AI-SC, RPB was 1.3+/-2.5 and 4.1+/-4.7 cubs, respectively. RPB and the percentage of vixens that weaned cubs were lower in NB-PC and NB-PE than in AI-PC. In AI groups, both RPB and RPM at weaning tended to be better in singly-housed than in pair-housed blue fox vixens. No statistically significant differences were found between AI groups in the percentage of vixens without oestrus, barren vixens, vixens that lost all cubs or weaned at least one cub. Only in one pair (AI-PC) both vixens displayed communal breeding and weaned cubs. The present results show that vixens in traditional breeding system had better RPM than in any of the studied pair-housing systems. The current blue fox population has been effectively selected for cage-breeding and artificial reproduction, and therefore blue foxes generally reproduce well with artificial insemination after careful detection of oestrus in traditional breeding conditions.     

Ultrastructure of in-vivo fertilization in superovulated cattle

Heifers were induced to superovulate by treatment with PMSG or FSH. Subsequently, oestrus was induced with prostaglandins and artificial insemination was performed. Ova were collected from the oviducts and their ultrastructural features were related to an estimated time of ovulation based on the time of the LH peak. With the insemination schedule used, the estimated time of ovulation defined the time at which fertilization was expected to occur. The ova were characterized as unfertilized, fertilized or possibly fertilized, and a sequence of nuclear and cytoplasmic changes associated with fertilization was revealed. Within 4 h after the estimated time of ovulation formation of the female and male pronucleus was initiated, and at 5-7 h swelling of the pronuclei occurred. At 19 h the pronuclei were closely apposed and synkaryosis was seen, and at 23 h the first two-cell stage was obtained. Within 2-3 h after the estimated time of ovulation cortical granule release, development of conspicuous Golgi complexes, and transformation of the smooth endoplasmic reticulum occurred. At approximately 7 h parallel arrays of annulate lamellae appeared. In one third of the unfertilized ova deviant oocyte maturation was noticed.     

Cytogenetic analysis of mouse oocytes after experimental induction of follicular overripening

Graafian follicle overripening was induced in (1) adult mice by inhibiting the ovulatory discharge of gonadotrophins with antibodies to LH-RH and (2) immature mice by injection of PMSG to promote follicular maturation before the neuroendocrine system was competent to produce an ovulatory stimulus. The numbers of follicles capable of meiotic maturation after exogenous LH were sharply reduced during the period of overripening and there was a corresponding increase in the proportion of cystically enlarged follicles, many of which were undergoing atresia. Freshly ovulated ova were collected after delaying ovulation for 2 days and prepared for cytogenetic study of metaphase chromosomes. The incidence of non-disjunction and other errors was indistinguishable from that of ova collected after spontaneous ovulation during 4- or 5-day cycles.     

Altered meiotic regulation in oocytes from diabetic mice

In the present study, we have utilized a streptozotocin-induced diabetic mouse model to examine how the diabetic condition and different glucose concentrations affect several parameters of reproductive physiology. We report that oocyte maturation is altered under all experimental conditions examined. In cumulus cell-enclosed oocytes (CEO) from diabetic mice, spontaneous maturation was accelerated but the FSH-mediated delay of spontaneous maturation was suppressed. Higher glucose levels in the culture medium suppressed spontaneous maturation but did not influence the transient arrest mediated by FSH. Meiotic arrest in CEO by hypoxanthine and dibutyryl cAMP (dbcAMP) was less effective at higher glucose concentrations. In addition, both FSH-induced maturation in vitro and hCG-induced maturation in vivo were reduced by the diabetic condition. The ovulation rate was lowered by about 50% in diabetic mice and fewer ovulated ova had reached metaphase II. Despite the decreased number of ova at metaphase II, in vitro cultures showed the oocytes were capable of completing meiotic maturation at control levels. Insulin treatment reversed the detrimental effects of diabetes on meiotic induction, ovulation, and completion of meiotic maturation. Cultures of pronuclear-staged embryos confirmed a negative effect of diabetes and hyperglycemia on development to the blastocyst stage. These data suggest that defects in meiotic regulation brought about by the diabetic condition are due to decreased communication between the somatic and germ cell compartments, and it is concluded that such conditions may contribute to postfertilization developmental abnormalities.     

Ultrastructure of canine oocytes during in vivo maturation

The aim of the present study was to describe the canine oocyte ultrastructural modifications during in vivo maturation, with precise reference to the timing of the LH surge and of ovulation. Twenty-five bitches were ovariectomized at specific stages between the onset of proestrus and the fifth day post-ovulation: 65 oocytes were observed by transmission electron microscopy (TEM), either before the LH surge (n = 10), between the LH surge and ovulation (n = 12) or after ovulation (n = 43). Prior to the LH surge, the oocyte nucleus had already begun its displacement to the vicinity of the oolemma and reticulated nucleoli were infrequent. The cytoplasm showed signs of immaturity (few organelles preferentially located in the cortical zone, "mitochondrial cloud", scarce cortical granules). The LH surge was immediately followed by cumulus expansion but the ovulation occurred 2 days later. Retraction of the transzonal projections and the meiotic resumption occurred after another 3 days (5 days after the LH peak). The ovulation was then followed by gradual cytoplasmic modifications. Nucleoli re-assumed a reticulated aspect around 24 hr post-ovulation. From 48 hr post-ovulation mitochondria and SER were very numerous and evenly distributed. In conclusion canine oocyte maturation began prior to the LH surge and no cytoplasmic or nuclear modifications followed immediately the LH surge and ovulation. This study suggests that two distinct signals are needed for the final in vivo maturation: one prior to the LH surge (to induce maturation) and another one, around 3 days post-ovulation (to induce meiotic resumption).     

Fertilization of rat eggs in vitro at various times before and after ovulation with special reference to fertilization of ovarian oocytes matured in culture.

Oocytes recovered at various times from immature rats treated with PMSG and HCG were incubated with capacitated epididymal spermatozoa of mature rats. In the presence of follicular cells, sperm penetration was not observed 4 hr after incubation in the oocytes at stages from the intact germinal vesicle to the chromatin mass, but 7 to 55% of oocytes were penetrated at stages from the condensed germainal vesicle to metaphase II. After the removal of follicular cells, 15 to 72% of the oocytes at any stage were penetrated. After further incubation for 15 hr, the proportion of penetrated oocytes increased from 8 to 98% from early to late stages and that of penetrated oocytes with a male and female pronucleus increased from 9 to 100% as maturation progressed. Although the average number of spermatozoa/oocyte was not correlated with its maturation, transformation of the sperm head into a male pronucleus was retarded or failed, especially in the younger oocytes. Following incubation in a defined medium for 13 hr, 85% of oocytes at the intact germinal vesicle stage matured to the stage of the first polar body formation, but only 18 to 22% of these mature oocytes were penetrated by spermatozoa and only a few of the penetrated oocytes cleaved into normal two-cell eggs. When eggs recovered from oviducts 14 to 20 hr after ovulation were exposed to capacitated spermatozoa, the proportion of penetrated eggs (86 to 98%) and that of polyspermic eggs (11 to 27%) were not related to the ages of the eggs, but failure of transformation of the sperm head and the proportion of abnormal eggs increased 14 to 20 hr after ovulation.     

Pronuclear formation in starfish eggs inseminated at different stages of meiotic maturation: correlation of sperm nuclear transformations and activity of the maternal chromatin

Changes in sperm nuclei incorporated into starfish, Asterina miniata, eggs inseminated at different stages of meiosis have been correlated with the progression of meiotic maturation. A single, uniform rate of sperm expansion characterized eggs inseminated at the completion of meiosis. In oocytes inseminated at metaphase I and II the sperm nucleus underwent an initial expansion at a rate comparable to that seen in eggs inseminated at the pronuclear stage. However, in oocytes inseminated at metaphase I, the sperm nucleus ceased expanding by meiosis II and condensed into chromosomes which persisted until the completion of meiotic maturation. Concomitant with the formation and expansion of the female pronucleus, sperm chromatin of oocytes inseminated at metaphase I enlarged and developed into male pronuclei. Condensation of the initially expanded sperm nucleus in oocytes inseminated at metaphase II was not observed. Instead, the enlarged sperm nucleus underwent a dramatic increase in expansion commensurate with that taking place with the maternal chromatin to form a female pronucleus. Fusion of the relatively large female pronucleus and a much smaller male pronucleus was observed in eggs fertilized at the completion of meiotic maturation. In oocytes inseminated at metaphase I and II, the male and female pronuclei, which were similar in size, migrated into juxtaposition, and as separate structures underwent prophase. The chromosomes in each pronucleus condensed, intermixed, and became aligned on the metaphase palate of the mitotic spindle in preparation for the first cleavage division. These observations demonstrate that the time of insemination with respect to the stage of meiotic maturation has a significant effect on sperm nuclear transformations and pronuclear morphogenesis.     

Fine structural localization of alkaline phosphatase in rat ovum during cleavage

On a submicroscopic level alkaline phosphatase activity was demonstrated by cytochemical methods in all stages of segmenting rat ova under survey, i.e. in the unfertilized and fertilized ovum, in the two-, four- and eight-cell stages and in the blastocyst. The reaction product was present in some cytoplasmic organelles as well as on cell membranes. A considerable number of cytoplasmic organelles with alkaline phosphatase activity was found in all stages from the one-cell up to the eight-cell stage. The reaction product was deposited in the tubules and vesicles of the smooth endoplasmic reticulum, in the nuclear envelope and in the Golgi complex as well. Some multivesicular bodies, autophagic vacuoles and majority of residual bodies out of the secondary lysosomes showed enzymatic activity. In the multicellular stages no significant differences were observed between the individual blastomeres in the incidence and distribution of the alkaline phosphatase activity. On the blastocyst-stage was found a low incidence of enzymatically active cytoplasmic organelles. Alkaline phosphatase activity was demonstrated in some minute vesicles below the cell membrane and in some secondary lysosomes. No essential differences were found between the cells of the embryoblast and the cells of the trophoblast in the incidence of enzymatically active structures. In the one-cell stage the activity of alkaline phosphatase was present on the cell membrane only sporadically, in the two- and four-cell stages enzymatic activity was found in this localization in a third of all specimen. In the eight-cell stage alkaline phosphatase activity was demonstrated on the cell membranes of all blastomeres. In the blastocyst the reaction product was deposited regularly on the membranes of the trophoblastic cells turned towards the zone pellucida, frequently on membranes of mutual tactile cells of the trophoblast and the embryoblast and only sporadically on cell membranes limiting the blastocyst cavity.     

Early embryonic development and in vitro culture of in vivo produced embryos in the farmed European polecat (Mustela putorius)

Early embryonic development and in vitro culture of in vivo produced embryos in the farmed European polecat (Mustela putorius) was investigated as a part of an ex situ conservation program of the endangered European mink (Mustela lutreola), using the European polecat as a model species. The oestrus cycles of 34 yearling polecat females were monitored by visual examination of the vulval swelling and, to induce ovulation, the females were mated once daily on two consecutive days. Sixteen yearling males were used for mating. The females were humanely killed 3-14 days after the first mating and the uteri and oviducts were collected for embryo recovery. Uterine and oviductal flushings yielded a total number of 295 embryos, representing developmental stages from the 1-cell stage to large expanded and hatched blastocysts. On Day 3 after the first mating, only 1-16-cell stage embryos were recovered. Between Days 4 and 6 after the first mating, 1-16-cell stage embryos and morulae were found. The first blastocysts were recovered on Day 7 after the first mating. The first implanted blastocysts were detected on Day 11 after the first mating. A total number of 85 embryos were in vitro cultured after recovery. Blastocyst production rates for in vitro cultured 1-16-cell stage embryos and for morulae/compact morulae were 68 and 84%, respectively. For all cultured embryos, the hatching rate was 15%. The in vitro culture requirements for the preimplantation embryos of the farmed European polecat remain to be determined before further utilization of the technique.     

Influence of mating on ovarian follicle development in Triatoma infestans (Klug, 1834).

This work examines the influence of mating on ovarian follicle development in Triatoma infestans. The observations were carried out on both virgin and mated females, which were killed at various times after their emergence. There was no difference in the ovarian development of both experimental groups during the first gonadotrophic cycle. By the 7th day mated females as well as virgin females showed vitellogenic oocytes. The coriogenesis and ovulation process began on the 13th day after imaginal moulting. However we could observe that egg-laying was dependent on mating. Mated females laid eggs whereas virgin females did not lay eggs. However ovarian production was significantly greater in the mated females. It is suggested that in T. infestans mating stimulates egg-laying but it does no influence the oogenesis and ovulation process.     

Full-term development of bovine follicular oocytes matured in culture and fertilized in vitro

Follicular oocytes were cultured for 28h in vitro and 91% of the oocytes reached the the second metaphase in culture. The penetration rate after insemination in vitro using frozen-thawed spermatozoa was 81%. After cultivation for 48h in vitro, 18% of the in vitro fertilized oocytes developed to the three- to four-cell stages and 21% of these developed to the six- to eight-cell stages. Following in vivo culture in the rabbit oviduct, 18% of six- to eight-cell and 5% of three- to four-cell embryos developed to the blastocyst stage. To confirm the full developmental competence, 11 blastocysts were transferred to recipient cows, and six (55%) cows became pregnant or delivered calves.     

The pattern of reproduction in female Columbian black-tailed deer, Odocoileus hemionus columbianus.

Ovarian cycles and the pattern of reproduction in female black-tailed deer in British Columbia were ascertained largely through examination of the ovaries from 444 females. Cyclic development and degeneration of single follicles of ovulatory size occurred several weeks before first ovulation. As the breeding season approached, a second or third large follicle developed in each cycle but in 48% of adult females the follicles were at different stages of maturation. Those failing to rupture at first ovulation luteinized 1 to 2 days thereafter. The first ovulation of the season, in November, never resulted in a lasting pregnancy even though some ova were penetrated by spermatozoa and began to cleave. First ovulation was apparently 'silent' in five of seven females for their ova lacked spermatozoa. Of sixty-one pregnant females, fifty-nine conceived at second ovulation; the other two conceived at subsequent ovulations more widely spaced than the 8- to 9-day interval between first and second ovulations. The synchrony of ovulatory cycles among adult females was such that half of them ovulated for the second time in a span of 7 or 8 days. Primary CL that formed after first ovulation grew to an average maximum volume of only about 45 mm3, whereas those originating at second ovulation grew to twice that size within 5 to 8 days. First generation CL shrank from 35 mm3 to 10 mm3 within 2 days. They disappeared within 18 months but corpora albicantia persisted for the life of the female. The possible ecological significance of the reproductive pattern is discussed.     

Changes in the cytologic and electrophysiologic parameters of rat oocytes in response to prostaglandins F2 alpha and E2

Influence of prostaglandins F-2 alpha and E-2 on ovulation character and on the state of the ovulated gametes has been studied in rats at the preovulatory period (the preparations have been injected for 3 days, beginning from the 1st day of early proestrus). Under the effect of prostaglandins the amount of postovulated ova decreases, especially in the animals given prostaglandin E-2. Inhibition of ovulation in these animals is accompanied with an increased part of hyperpolarized ova, appearance of some signs of degeneration in the group of depolarized oocytes and absence of oocytes with polarized membrane at the metaphase I stage. These facts can be interpreted as desynchronization of maturation processes and ova ovulation. Mechanisms of the changes revealed in the ovulatory process and the state of gametes under the prostaglandins effect are discussed.     

Nucleolar ultrastructure in bovine nuclear transfer embryos

Nuclear transfer experiments in mammals have attempted to reprogram a donor nucleus to a state equivalent to the zygotic one. Reprogramming of the donor nucleus is, among other features, indicated by a synthesis of ribosomal RNA (rRNA). The initiation of rRNA synthesis is simultaneously reflected in nuclear morphology as a transformation of the nucleolus precursor body into a functional rRNA synthesising nucleolus with a characteristic ultrastructure. We examined nucleolar ultrastructure in bovine in vitro produced (control) embryos and in nuclear transfer embryos reconstructed from a MII phase (nonactivated) or S phase (activated) cytoplasts. Control embryos were fixed at the two-, four-, early eight- and late eight-cell stages; nuclear transfer embryos were fixed at 1 and 3 hr post fusion and at the two-, four-, and eight-cell stages. Control embryos possessed a nucleolar precursor body throughout all three cell cycles. In the eight-cell stage embryo, a primary vacuole appeared as an electron lucid area originating in the centre of the nucleolar precursor body. In nuclear transfer embryos reconstructed from nonactivated cytoplasts, the nuclear envelope was fragmented or completely broken down at 1 hr after fusion and, by 3 hr after fusion, it was restored again. At this time, the reticulated fibrillo-granular nucleolus had an almost round shape. The nucleolar precursor body seen in the two-cell stage nuclear transfer embryos consisted of intermingled filamentous components and secondary vacuoles. A nucleolar precursor body typical for the two-cell stage control embryos was never observed. None of the reconstructed embryos of this group reached the eight-cell stage. Nuclear transfer embryos reconstructed from activated cytoplasts, in contrast, exhibited a complete nuclear envelope at all time intervals after fusion. In the two-cell stage nuclear transfer embryo, the originally reticulated nucleolus of the donor blastomere had changed into a typical nucleolar precursor body consisting of a homogeneous fibrillar structure. A primary vacuole appeared in the four-cell stage nuclear transfer embryos, which was one cell cycle earlier than in control embryos. Only nuclear transfer embryos reconstructed from activated cytoplasts underwent complete remodelling of the nucleolus. The reorganisation of the donor nucleolar architecture into a functionally active nucleolus was observed as early as in the four-cell stage nuclear transfer embryo. These ultrastructural observations were correlated with our autoradiographic data on the initiation of RNA synthesis in nuclear transfer embryos.