The mutual benefits of research in wild animal species and human-assisted reproduction.

Studying the reproductive biology of wild animal species produces knowledge beneficial to their management and conservation. However, wild species also share intriguing similarities in reproductive biology with humans, thereby offering alternative models for better understanding the etiology of infertility and developing innovative treatments. The purpose of this review is to raise awareness in different scientific communities about intriguing connections between wild animals and humans regarding infertility syndromes or improvement of fertility preservation. The objectives are to (1) highlight commonalities between wild species and human fertility, (2) demonstrate that research in wild species-assisted reproductive technologies can greatly enhance success in human reproductive medicine, and (3) recognize that human fertility preservation is highly inspiring and relevant to wild species conservation. In addition to having similar biological traits in some wild species and humans, the fact of sharing the same natural environment and the common needs for more options in fertility preservation are strong incentives to build more bridges that will eventually benefit both animal conservation and human reproductive medicine.

Oogenesis: Prospects and challenges for the future.

Oogenesis serves a singular role in the reproductive success of plants and animals. Of their remarkable differentiation pathway what stands out is the ability of oocytes to transform from a single cell into the totipotent lineages that seed the early embryo. As our understanding that commonalities between diverse organisms at the genetic, cellular and molecular levels are conserved to achieve successful reproduction, the notion that embryogenesis presupposes oogenesis has entered the day-to-day parlance of regenerative medicine and stem cell biology. With emphasis on the mammalian oocyte, this review will cover (1) current concepts regarding the birth, survival and growth of oocytes that depends on complex patterns of cell communication between germ line and soma, (2) the notion of "maternal inheritance" from a genetic and epigenetic perspective, and (3) the relative value of model systems with reference to current clinical and biotechnology applications.

One-step versus two-step culture of mouse preimplantation embryos: is there a difference?

BACKGROUND: A comparison has been made of the development of mouse zygotes in either one-step or two-step culture systems. METHODS: Embryo culture, blastocyst cell counts and embryo transfer were done. RESULTS: No significant differences were observed in the proportions of blastocysts, rates of hatching, numbers of cells in the inner cell mass (ICM) and trophectoderm (TE) that developed in protocols: one-step culture in potassium-enriched simplex optimized medium supplemented with glucose and amino acids (KSOMg(AA)), two-step culture in KSOMg(AA)/KSOMg(AA), and two-step culture in G1.2/G2.2. No gross abnormalities were observed in the fetuses that developed from zygotes in the one-step protocol using KSOMg(AA) and a two-step protocol using G1.2/G2.2. The body weights of these two groups of fetuses were not significantly different and no developmental abnormalities were observed. No significant differences were observed in the proportions of blastocysts, rates of hatching, numbers of cells in the ICM and TE that developed in protocols: one-step culture in KSOMg(AA), two-step culture in KSOMg(AA)/KSOMg(AA), and two-step culture in DM2/DM1. EDTA is not toxic to the initial cleavage stages of development at a concentration of 0.01 mmol/l in KSOMg(AA). CONCLUSIONS: Two-step culture protocols are sufficient for the support of preimplantation mouse development in vitro but they are not necessary.

Mouse embryo development following IVF in media containing either L-glutamine or glycyl-L-glutamine.

BACKGROUND: The development of the mouse zygote following fertilization in vitro in a KSOM-type medium containing either L-glutamine or glycyl-L-glutamine has been examined, and compared with the development of mouse zygotes produced by natural fertilization. METHODS: Mouse IVF, embryo culture and embryo transfer. RESULTS: Fertilization rates, development to the blastocyst stage, implantation rate, gross fetal development and fetal body weight are not different in a KSOM-type medium containing either L-glutamine or glycyl-L-glutamine. No evidence of abnormal fetal development, such as exencephaly, was observed. The replacement of L-glutamine with glycyl-L-glutamine favoured the development of relatively more inner cell mass cells than trophectoderm cells, and reduced the numbers of pyknotic and fragmented nuclei in the blastocysts that developed in vitro. CONCLUSIONS: There is no evidence that the presence of glutamine in the medium used for IVF influences significantly the subsequent development of the zygote. Replacing glutamine with glycyl-L-glutamine may be advantageous.

Evidence that glucose is not always an inhibitor of mouse preimplantation development in vitro.

A factorial experimental design was used to examine the effects of 16 combinations of four concentrations of glucose (0.20, 0.60, 1.8, 5.4 mmol/l) and four concentrations of potassium dihydrogen phosphate (KH(2)PO(4); 0.05, 0.15, 0.45, 1.35 mmol/l) on the development in vitro of outbred CF1 mouse zygotes. Three responses were measured: (i) the number of zona-enclosed blastocysts; (ii) the number of blastocysts that started to hatch; and (iii) the total cell counts in the blastocysts. General linear modelling was used to estimate the most parsimonious two-dimensional concentration-response surfaces that represent the three responses to the different concentrations of glucose and KH(2)PO(4). There were no significant interactions between the effects of glucose and KH(2)PO(4) in all cases. Thus, the effects of glucose and phosphate are independent. No significant effects of glucose on blastocyst formation and the initiation of hatching were observed. Increasing the concentration of KH(2)PO(4) inhibited slightly (

IVF of mouse ova in a simplex optimized medium supplemented with amino acids.

The addition of amino acids to a modified simplex optimized medium (mKSOM) did not increase the percentage of blastocysts that develop from CF1 mouse ova fertilized in vitro. In contrast, the percentage of blastocysts that began to hatch and the number of cells in these blastocysts, particularly in the inner cell mass, was increased. The added amino acids also supported the development of a more organized extracellular matrix in the same blastocysts. The results suggest that zygotes produced in amino acid-supplemented mKSOM have a greater developmental potential, perhaps developing at a faster rate, than zygotes produced in mKSOM. This enhanced developmental potential may be caused by the alleviation of osmotic stress on the ova and zygotes by the amino acids that are osmolytes. The fertilization of human ova in vitro may benefit from the inclusion of free amino acids in the fertilizing medium. The availability of a medium that can be used to support both IVF and preimplantation development in the mouse is likely to benefit the recovery of mouse strains from cryopreserved spermatozoa.

Amino acids and preimplantation development of the mouse in protein-free potassium simplex optimized medium.

Development of outbred CF1 mouse zygotes in vitro was studied in a chemically defined, protein-free medium both with and without amino acids. The addition of amino acids to protein-free potassium simplex optimized medium (KSOM) had little effect on the proportion of embryos that developed at least to the zona-enclosed blastocyst stage. In contrast, amino acids stimulated very significantly, in a dilution-dependent way, the proportion of blastocysts that at least partially or completely hatched. Amino acids also stimulated cell proliferation in both the trophectoderm and inner cell mass (ICM) cells, at rates that favored proliferation of cells in the ICM; had no effect on the incidence of cell death (oncosis or apoptosis); and improved development of the basement membranes, which form on the blastocoelic surface of the trophectoderm and between the primitive endoderm and the primitive ectoderm. Thus, KSOM, supplemented with amino acids but containing no protein supplements, supports development of a newly fertilized ovum to the late blastocyst stage, in which its normal, three-dimensional structure is preserved and in which the ICM has been partitioned into the primitive ectoderm and primitive endoderm.

Polyvinyl alcohol and amino acids as substitutes for bovine serum albumin in culture media for mouse preimplantation embryos.

The effect of replacing bovine serum albumin (BSA) in a simple defined medium (KSOM) with polyvinyl alcohol (PVA) and/or amino acids on the percentages of mouse zygotes that develop to at least the blastocyst stage and that hatch at least partially or completely is reported. Blastocysts could form when BSA was replaced with only PVA, but at a moderately reduced rate; however, partial hatching, and hence complete hatching, were severely impaired when BSA was replaced with only PVA. The substitution of BSA with amino acids alone resulted in a high rate of blastocyst formation and moderate impairment of hatching. The addition of PVA to BSA-free KSOM supplemented with amino acids had no extra effect. BSA had significant effects when added to BSA-free KSOM supplemented with amino acids. The BSA caused a significant increase in the rate of partial hatching, and may even have had a small effect on the rate of blastocyst formation. The results also showed that glucose, at a high concentration of 5.56 mM, does not inhibit the development of mouse zygotes to hatched blastocysts when cultured in KSOM supplemented with amino acids.

Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis.

THE D-type cyclins (D1, D2 and D3) are critical governors of the cell-cycle clock apparatus during the G1 phase of the mammalian cell cycle. These three D-type cyclins are expressed in overlapping, apparently redundant fashion in the proliferating tissues. To investigate why mammalian cells need three distinct D-type cyclins, we have generated mice bearing a disrupted cyclin D2 gene by using gene targeting in embryonic stem cells. Cyclin D2-deficient females are sterile owing to the inability of ovarian granulosa cells to proliferate normally in response to follicle-stimulating hormone (FSH), whereas mutant males display hypoplastic testes. In ovarian granulosa cells, cyclin D2 is specifically induced by FSH via a cyclic-AMP-dependent pathway, indicating that expression of the various D-type cyclins is under control of distinct intracellular signalling pathways. The hypoplasia seen in cyclin D2(-/-) ovaries and testes prompted us to examine human cancers deriving from corresponding tissues. We find that some human ovarian and testicular tumours contain high levels of cyclin D2 messenger RNA.

Chromatin organization, meiotic status and meiotic competence acquisition in mouse oocytes from cultured ovarian follicles.

Changes in chromatin organization, meiotic status and the development of meiotic competence in oocytes retained within mouse ovarian follicles from day 0 to day 6 in culture were examined. The effects of exposure for 24 h to human luteinizing hormone (hLH) during the last day in culture was also determined. Preantral follicles from 22- to 24-day-old (prepubertal) mice develop antra and undergo significant growth from day 0 to day 4 in culture, after which the growth rates slow. The statistical significance of meiotic progression was examined using exact logistical regression analysis, which is particularly useful when the data are sparse and unbalanced. The transition from rimmed to unrimmed germinal vesicle stages was found to occur between day 2 and day 4 of follicle culture and was not influenced by exposure to hLH. Treatment with hLH caused a significant increase in the proportion of intrafollicular oocytes resuming meiosis. Assays of meiotic competence performed in vitro in oocytes retrieved from cultured follicles demonstrated that the transition from an unrimmed to a rimmed state is closely coincident with the acquisition and expression of meiotic competence. Forty-six per cent of competent oocytes from follicle cultures at day 3 progressed to metaphase II. These results indicate that the follicle culture system used in these studies supports the transformation of enclosed oocytes from a precompetent to a competent state and can maintain meiotic arrest for up to 6 days in culture. However, an increasing proportion of oocytes exhibit abnormal meiotic progression with continued follicle culture beyond 4 days.

Investigations into the control of litter size in swine: I. Comparative studies on in vitro development of Meishan and Yorkshire preimplantation embryos.

Three experiments were conducted to examine the in vitro development of preimplantation embryos from the prolific Chinese Meishan pig. Experiment 1 was conducted to assess whether Meishan embryos would develop in vitro and retain their viability, whereas Exp. 2 and 3 examined the developmental pattern of Meishan embryos. In all three experiments, Yorkshire embryos served as a contemporary comparison. Ovulation and embryo recovery rates were not different between Meishan and Yorkshire gilts. Meishan embryos cultured for 96 h were capable of establishing pregnancies. The number of cell nuclei present after 144 h of culture was lower (P < .01) for Meishan than for Yorkshire blastocysts. Meishan preimplantation embryos exhibited a slower (P < .02) in vitro rate of development from the four-cell to the compact morula stage than did Yorkshire embryos. Early blastocysts from Meishan gilts, although morphologically similar in size, contained fewer (P < .06) cells than did their counterpart Yorkshire embryos. These data demonstrate that Meishan embryos develop more slowly and contain fewer cells than do Yorkshire embryos. This differing developmental pattern of Meishan preimplantation embryos, if similar to that previously reported in miniature swine and mice, may relate to increased embryo survival.

In vitro development of ovine embryos in CZB medium.

One- to four-cell embryos were collected from multiparous crossbred ewes and were cultured in vitro for 120 hours in CZB medium. A 2x2 factorial treatment arrangement was used to examine the effects of glucose and ethylenediaminetetraacetic acid (EDTA) on in vitro embryo development. The embryos were examined every 12 hours, and all of the embryos were stained with a DNA-specific fluorochrome after the 120-hour evaluation to enable the counting of cell nuclei. Embryo development was analyzed for cleavage beyond 16 cells as well as for cleavage to at least the compact morula stage based upon both the 120-hour morphological evaluation and nuclear counts. Forty-eight percent of the embryos passed through the in vitro developmental block (i.e., cleaved beyond 16 cells), and 26% developed to 30 or more cells. Neither EDTA nor glucose affected in vitro embryo development based on the nuclear counts.