R.F. Seamark

The production of unusually large offspring following embryo manipulation: Concepts and challenges

Abstract Embryo culture, nuclear transfer and asynchronous embryo transfer are procedures that can result in the birth of larger than normal offspring. Some birthweights fall well outside the upper limit of the normal population although a minority of offspring is usually affected. Adult liveweights appear not to be affected and the birthweight trait appears not to be heritable although definitive studies have not been reported. Preliminary evidence indicates that cell lineage differentiation in the manipulated embryo is altered resulting in preferential allocation of cells to the trophectoderm. Consequently, a larger than normal placenta develops resulting in aberrant fetal growth and development. It is postulated that this disturbance results from environmentally-induced changes in the regulation of early gene expression. Disruptions to nucleocytoplasmic interactions and to maternal — embryonic signalling are implicated in the phenomenon. The production of large offspring after embryo manipulation casts new perspectives on the roles of reproductive technology in both livestock and human reproduction. However, an understanding of the underlying cellular mechanisms should lead to improved procedures for the handling and manipulation of embryos.

IN VITRO CULTURE OF SHEEP EMBRYOS WITHOUT CO-CULTURE : SUCCESSES AND PERSPECTIVES

Abstract The finding that zygotes (one-cell embryos) from several livestock species can be routinely cultured in relatively simple media to the blastocyst stage questions the nature of the environment provided by the oviducts for the development of early stage embryos. In this paper, we review recent findings on the development of sheep embryos in simple media without co-culture. Evidence indicates that embryos are relatively insensitive to changes in the composition of media and that zygotes can develop to blastocysts at rates equal to or higher than those obtained in vivo. However, in vitro culture is associated with several developmental abnormalities. These include cytoplasmic fragmentation, early time of blastocoele formation and a reduced number of nuclei per blastocyst. Viability of embryos (to Day 50 of pregnancy) after 5 days of culture was reduced compared with embryos cultured in vivo (48.2% vs. 59.4%, P

Granulocyte-Macrophage Colony-Stimulating Factor Promotes Glucose Transport and Blastomere Viability in Murine Preimplantation Embryos

Abstract Granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion from epithelial cells lining the female reproductive tract is induced during early pregnancy by ovarian steroid hormones and constituents of seminal plasma. In this study we have investigated the influence of GM-CSF on development of preimplantation mouse embryos. Blastocyst-stage embryos were found to specifically bind 125I-GM-CSF and analysis of GM-CSF mRNA receptor expression by reverse transcriptase-polymerase chain reaction indicated expression of the low-affinity α subunit of the GM-CSF receptor, but not the affinity-converting β subunit (βc), or GM-CSF ligand. GM-CSF receptor mRNA was present in the fertilized oocyte and all subsequent stages of development, and in blastocysts it was expressed in both inner cell mass and trophectoderm cells. In vitro culture of eight-cell embryos in recombinant GM-CSF accelerated development of blastocysts to hatching and implantation stages, with a maximum response at a concentration of 2 ng/ml (77 pM). Blastocysts recovered from GM-CSF-null mutant (GM−/−) mice on Day 4 of natural pregnancy or after superovulation showed retarded development, with the total cell number reduced by 14% and 18%, respectively, compared with GM+/+ embryos. Blastocysts generated in vitro from two-cell GM−/− and GM+/+ embryos were larger when recombinant GM-CSF was added to the culture medium (20% and 24% increases in total cell numbers in GM+/+ and GM−/− blastocysts, respectively). Incubation of blastocysts with recombinant GM-CSF elicited a 50% increase in the uptake of the nonmetabolizable glucose analogue, 3-O-methyl glucose. In conclusion, these data indicate that GM-CSF signaling through the low-affinity GM-CSF receptor in blastocysts is associated with increased glucose uptake and enhanced proliferation and/or viability of blastomeres. Together, the findings implicate a physiological role for maternal tract-derived GM-CSF in targeting the preimplantation embryo, and suggest that defective blastocyst development contributes to compromised pregnancy outcome in GM-CSF-null mutant mice.

Pregnancies and live birth from in vitro fertilization of calf oocytes collected by laparoscopic follicular aspiration

Oocytes were recovered by laparoscopic aspiration from 3- to 8-week-old calves treated with follicle-stimulating hormone (FSH) followed by human chorionic gonadotropin (hCG) to induce follicular growth and oocyte maturation in vivo. Most of the recovered oocytes either had resumed meiotic maturation at the time of aspiration or were competent to undergo maturation during subsequent culture in vitro. Oocytes matured in vivo following FSH and hCG treatment underwent in vitro fertilization (70%) at rates not significantly different from those of control oocytes recovered from adult cow ovaries at abattoirs and matured in vitro (75%). Calf oocytes that were immature at aspiration exhibited lower fertilization rates after in vitro maturation (36%) but their rate of development to morulae and blastocysts did not differ from that of mature oocytes at aspiration. A total of 91% of the zygotes produced from calf oocytes developed to morula and 27% to blastocyst stages during 6 days of culture. The proportion developing to morulae was significantly higher (P<0.05) than that observed for zygotes resulting from in vitro maturation and fertilization of oocytes recovered from cow ovaries obtained at an abattoir and processed concomitantly (59% to morulae and 18% to blastocysts). Morulae or blastocysts developed from oocytes from 5 to 6-week-old calves, when transferred to synchronized recipient heifers, resulted in 2 confirmed pregnancies, one of which produced a single full-term live calf. The ability to produce embryos from oocytes recovered from newborn or prepubertal calves offers the potential for markedly reducing the generation interval in cattle, thereby substantially accelerating the rate of genetic gain that can be achieved through embryo transfer.

MORPHOLOGICAL AND FUNCTIONAL RELATIONS OF GRAAFIAN FOLLICLE GROWTH TO OVULATION IN WOMEN USING ULTRASONIC, LAPAROSCOPIC AND BIOCHEMICAL MEASUREMENTS

The daily growth rates of ovarian follicles were recorded ultrasonically for five days until ovulation in 56 spontaneously ovulating women and related to endocrine and clinical parameters. Over the 5‐day period, the average diameter of the follicle destined to ovulate increased from 12 to 23 mm, the second largest follicle from 6 to 12 mm, the third largest follicle from 5 to 9 mm and the fourth largest follicle from 4 to 8 mm. Similar but lesser growth rates occurred in the follicles in the contralateral ovary. Ovulation occurred within 24 hours of the luteinizing hormone (LH) peak, and the mean peak diameter of the ovulating follicle was 23.2±0.3 (SEM) mm, (range 18–29 mm) before ovulation, and subsequent luteal function was judged to be normal. Follicular growth was most closely correlated with increasing peripheral blood oestrogen levels. In 16 women who had a laparoscopy within 12 hours of the last ultrasound and following the LH peak, the mean diameter of the largest follicle as measured by ultrasound (23.6±0.4 mm) was similar to that measured at laparoscopy (22.8±0.4 mm) and estimated from the volume of follicular fluid aspirated (average 5.8±0.2 ml), 22.5 mm. The follicular fluid levels of progesterone were high on the day of the LH peak and blood progesterone levels had risen significantly indicating that luteinization of the dominant Graafian follicles had already occurred prior to ovulation. This study confirms that ultrasonic monitoring provides a reliable measure of follicular growth and allows studies correlating morphological changes with both normal and abnormal endocrine function of the human ovary.

Human luteal phase function following oocyte aspiration from the immediately preovular graafian follicle of spontaneous ovular cycles.

Human luteal phase function as evaluated by peripheral venous blood steroid levels does not appear to be impaired following the aspiration of follicular fluid together with a cumulus enclosed oocyte and a number of granulosal cells from the immediate preovular follicle in women having otherwise spontaneous ovular cycles. The day to day levels of luteinising hormone, oestradiol‐17β, 17α‐hydroxyprogesterone, progesterone and basal temperatures in 14 women who had their preovular follicle aspirated were compared with a control group of 28 spontaneously ovulating women. It was concluded that a carefully performed single aspiration of the contents of a preovular follicle, for the purpose of extra‐corporeal fertilisation of the mature oocyte, did not lead to impaired steroid function of the subsequent corpus luteum, although the prolactin levels were increased due to the effects of the relaxant anaesthetic and/or the laparoscopic procedure. A safe and simple laparoscopic procedure is also described, which is particularly suitable for women with a likelihood of extensive pelvic adhesions.

Gonadotropin stimulation regimens for follicular aspiration and in vitro embryo production from calf oocytes

Crossbred beef x dairy calves were randomly allocated at 3 wk of age to different gonadotropin treatment regimens for stimulation of follicle development and induction of oocyte maturation in vivo. Follicular responses were assessed laparoscopically, and oocytes were aspirated for assessment of maturational state or for in vitro fertilization (IVF) and culture to determine developmental capacity. Follicle-stimulating Hormone (FSH), administered in a single subcutaneous injection together with a low dosage of PMSG, was as effective as the same total dosage of FSH administered in 6 injections over a 3-d period. Without accompanying PMSG, this dose of FSH was ineffective in stimulating follicle development. The mean number of preovulatory follicles (> 5mm, with hyperemic appearance) doubled with each successive stimulation at 3-wk intervals, reaching 35 follicles per calf at 9 wk of age. Oocyte yields ranged from 55 to 81% of follicles aspirated, and did not differ significantly among age, FSH regimen and oocyte maturation stimulus. A combination of LH + FSH was more effective in stimulating cumulus cell expansion than LH by itself (73 vs 22% of recovered oocyte-cumulus cell complex (OCC) respectively; P<0.05). Of 33 unselected immature oocytes (cumulus unexpanded) subjected to in vitro maturation (IVM) and IVF, 30% developed to blastocysts during co-culture with bovine oviduct epithelial cells, which was not significantly different from 25% of 36 oocytes from adult ovaries which reached the blastocyst stage under similar conditions. The results indicate that follicle responses of calf ovaries to FSH stimulation increase progressively from 3 to 9 wk of age, and that oocytes recovered laparoscopically from these follicles produce blastocysts in culture at rates similar to oocytes from adult cattle ovaries collected at slaughter. The approach offers promise for embryo production from donor calves of superior genetic merit for embryo transfer, thereby enhancing the rate of genetic gain above that attainable by conventional breeding or by embryo transfer in adult cattle.

Cytokines in rodent reproduction and the cytokine-endocrine interaction

Insights derived from recent studies employing rodent models demonstrate that the synthesis of pluripotent cytokines is an important function of resident cells in the female reproductive tract. Through steroid hormone regulated secretion of these mediators, resident cells appear to coordinate the recruitment and action of leukocytes that are centrally implicated in the dramatic remodelling processes characteristic of reproductive events.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) targets myeloid leukocytes in the uterus during the post-mating inflammatory response in mice

Factors in seminal plasma elicit a surge of GM-CSF expression in uterine epithelial cells after mating in mice. This study investigates the nature of the endometrial cell populations targeted by epithelial GM-CSF. In quantitative RT-PCR studies, expression of the alpha-subunit of the GM-CSF receptor (GM-CSF-R) parallelled GM-CSF expression, being maximal during the 48 h period after mating and declining thereafter. Expression of mRNA encoding beta-common chain (AIC2B) also increased after mating and remained high until the time of embryo implantation on day 4 of pregnancy. Cells expressing GM-CSF receptors were identified in sections of uterus on the day after mating using 125I-GM-CSF, and were located predominantly in the endometrial stroma subjacent to the luminal epithelium, co-localising with abundant populations of myeloid leukocytes. Cells expressing GM-CSF receptor were identified as macrophages, granulocytes and putative dendritic cells by flow cytometric analysis using lineage and receptor subunit specific antibodies. Recombinant GM-CSF injected into the uterine lumen of ovariectomised mice was found to elicit a dose-dependant accumulation of macrophages and granulocytes in the endometrium, in a pattern of distribution comparable to that seen in uteri after natural mating. Together, these data indicate a role for epithelial cell-derived GM-CSF in mediating the recruitment and potentially in modifying the behaviour of uterine leukocytes during the post-mating inflammatory response in mice.

CHANGES IN LYMPHOCYTE FUNCTION DURING PREGNANCY

A gradual increase in spontaneous lymphocyte DNA synthesis was demonstrated in each trimester of pregnancy. Autoradiographic studies indicated that lymphocytes were primarily responsible for this activity. PHA‐induced lymphocyte transformation in both fetal calf serum and autologous serum was significantly reduced in the second and third trimesters of pregnancy. Spontaneous lymphocyte DNA synthesis was significantly reduced in patients with mild pre‐eclampsia. However, no significant differences were seen in patients with severe pre‐eclampsia in the third trimester of pregnancy compared with the normal control subjects. No evidence was adduced to implicate inhibitory humoral factors affecting the peripheral blood lymphocytes in pregnant patients in experiments in which washed lymphocytes were cultured in medium containing heterologous serum. In vitro experiments demonstrated that cortisol, progesterone and HPL caused a significant reduction in lymphocyte DNA synthesis, and HGH and HCG had a variable effect. However, only cortisol was regularly inhibitory at physiological concentrations. The progesterone effect was dose‐related, producing 90 per cent inhibition of activity at a concentration of 10 μg/ml. No synergism could be shown between HPL and progesterone on lymphocyte transformation. The increase in activity of circulating immunoreactive cells during pregnancy and its depression with the onset of pre‐eclampsia is discussed.