Multicellular living organisms and unmodified parts thereof and – Method of making a transgenic nonhuman animal – Via microinjection of a nucleus into an embryo – egg cell – or...
Reexamination Certificate
2001-04-09
2003-04-15
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of making a transgenic nonhuman animal
Via microinjection of a nucleus into an embryo, egg cell, or...
C800S008000, C800S017000
Reexamination Certificate
active
06548741
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the harvesting of porcine oocytes with improved developmental competence and to the generation of pigs by assisted reproduction strategies, including nuclear transfer including but not being limited to, genetically selected and/or modified animals.
BACKGROUND OF THE INVENTION
The female reproductive cycle begins with maturation and ovulation of the oocyte within the female reproductive tract. In normal sexual reproduction, fertilization of the oocyte by sperm also occurs in the reproductive tract. Assisted reproduction strategies, such as in vitro fertilization (IVF) and nuclear transfer, use oocytes that are harvested from the female animal at some point prior to fertilization and subsequently processed in vitro. Since the fertile life span of an oocyte is short, defining the optimal period within which to retrieve oocytes for reproductive strategies is invaluable, as is insight into how that period can be manipulated. Currently, assisted reproduction strategies use oocytes that are selected at random from a matured oocyte population. Typically, such oocyte populations are removed from the ovaries of slaughterhouse animals and matured in vitro, or are harvested from superovulated pigs and are thus matured in vivo.
There is little known of the factors that define the competence of an animal oocyte to develop following ovulation and/or maturation. Although developmental competence is positively correlated with the size of the follicles from which they are derived (Pavlok et al.,
Mol. Reprod. Dev.
35 233-243 (1993)), not all large follicles ovulate and in fact most undergo atresia, with concomitant loss of the oocyte within (Driancourt,
Theriogen
35 55-79 (1991)). A more likely factor is the requirement for a specific follicular steroid environment (Moor et al.,
JEEM
56 319-335 (1980)). In support of this studies in cattle and sheep have demonstrated follicular differences in growth and steroid hormone expression correlated with dominant and secondary waves of folliculogenesis (reviewed by Roche
Rev of Reprod
1 19-27 (1996)).
To overcome the hurdles imposed by the finite life and undefined characteristics of a developmentally competent oocyte two approaches have been taken. The first approach uses exogenous gonadotropins to induce (super) ovulation, and the second bypasses ovulation and recapitulates the maturation of oocytes in vitro under defined conditions (reviewed in Boland and Roche,
Mol. Reprod. Dev.
36 266-270 (1993)). Both methods improve the success of reproductive strategies by providing increased numbers of oocytes for subsequent insemination and/or manipulation. However, these methods do not guarantee optimal developmental competence of the oocytes that they provide. Superovulation of prepubertal sheep, pigs, and cattle still results in oocytes of diminished developmental competence compared with oocytes derived from post-pubertal animals (Wright et al.,
J. Anim. Sci.
42 912-917 (1976); Pinkert et al.,
J Reprod. Fert.
87 63-66 (1989); Seidel et al.,
Dairy Sci.
54 923-926 (1971)). In addition, oocytes matured in vitro as cumulus-oocyte-complexes are highly heterogeneous in their morphology and quality. The abundance of cumulus coverage surrounding oocytes has been positively correlated with their developmental competence following in vitro maturation (Leibried-Rutledge and First,
J. Anim. Sci.
48 76-86 (1979); De Loos et al.,
Gamete Res.
24 197-204 (1989); Blondin and Sirard,
Mol. Reprod. Dev.
41 54-62 (1995); Funahashi and Day,
J. Reprod. Fert. Suppl.
52 271-283 (1997)).
Previous studies in the pig have monitored ovulation by either slaughter, laparoscopy, or transcutaneous or transrectal ultrasound (Hunter
Res. Vet. Sci.
13 356-361 (1972); Brussow et al.,
Reprod. in Dom. Anim.
25 255-260 (1990); Weitze et al.,
Reprod. Dom. Anim.
25 61-67 (1990); Weitze et al
Zuchthyg.
24 40-42 (1989); Soede et al.,
Theriogen
38 653-666 (1992)). In the pig the timing of ovulation during the oestrus phase of the reproductive cycle is highly variable. For example, in spontaneously ovulating animals, timing may vary from between 10 to 85 hours after the onset of oestrus (reviewed by Soede and Kemp
J. Reprod. Fert. Suppl.
52 91-103 (1997)). This can be shortened by 5 to 14 hours following intracervical infusion of seminal plasma (Weitze et al.,
Reprod. Dom. Anim.
25 61-67 (1990)). Such infusion has no effect on the synchronized ovulatory response of gonadotropin treated gilts (Brussow et al.,
Reprod. Dom. Anim.
28 119-122 (1992)), which ovulate between 35 to 50 hours post stimulation depending on the gonadotropin used (reviewed by Kemp and Soede,
J. Reprod. Fertil. Suppl.
52 79-89 (1997)).
Seasonally reduced fertility in pigs, known as “summer infertility”, has been reported, although this effect is less pronounced than in wild pigs or other species such as the sheep (reviewed by Claus and Weiler
J. Reprod. Fert. Suppl.
33 185-197 (1985)). In sheep, variations in the duration of day length have been demonstrated as the regulatory stimulus controlling the pattern of seasonal reproduction. Effects of photoperiod are transduced from neuronal to endocrine signals by the stimulation of the pineal gland, which produces hormones such as melatonin that in turn stimulate gonadotropin release. Diurnal variations in porcine plasma melatonin have been described (Klupiec et al.,
J. Pineal Res.
22 65-74 (1997)). However, there is no evidence that melatonin or its precursors and metabolites are involved in regulating ovulation or reproductive performance (Foxcroft et al., in “Principles of pig science”, ed. DJA Cole, Nottingham University Press (1994)).
In general the mechanisms establishing ovulation rate in the pig are poorly understood. In the pig, growth and development of a follicle is dependent on pituitary gonadotropins from the time it acquires theca interna cells. The growth of selected preovulatory follicles in the pig is also associated with rapid atresia of smaller follicles (Foxcroft and Hunter,
J. Reprod. Fertil. Suppl.
33 1-19 (1985)). This is consistent with the existence of a dominant follicular wave suggested for monotocous species such as cattle. Recruitment of follicles from the antral follicle pool (and thus rescue from atresia) is also known to be induced by a specific pattern of episodic Lutenizing hormone release (reviewed by Kemp et al., “Control of Ovulation” in “Progress in Pig Science”, ed. Wisemean, Nottingham University Press (1988)).
A limited number of studies have attempted to examine the relationship between embryonic survivability and either the duration of ovulation or its early induction in the pig. Initial reports suggested that the first ovulating follicles resulted in the best developing embryos in a litter (Pope et al.,
Biol. of Reprod.
39 822-887 (1988); Xie et al.,
Biol. of Reprod.
43 236-240 (1990)). However, these results have been challenged by others (Soede et al.,
Theriogen
38 653-666 (1992); Soede and Kemp
Theriogen
39 1043-1053 (1993)). Early embryonic survivability has also been found to be unaffected by early induction of ovulation, induced by varying the timing of gonadotropin injections relative to each other (reviewed by Kemp et al., “Control of Ovulation” in “Progress in Pig Science”, ed. Wisemean, Nottingham University Press (1988)). There have been no reports suggesting improved embryonic survivability following delayed ovulation of the oocyte used to prepare the embryo. A single study has described the collection of porcine oocytes at later time points relative to injection of gonadotropin in which the oocytes are said to show an improved capacity to be activated and develop to the blastocyst stage following nuclear transfer. However, in this study the timing of ovulation was not recorded and nor was the stage in the diurnal cycle of the animal noted. The beneficial effect was attributed by the authors to ageing of the oocytes within the oviduct (Nagashima et al.,
Mol. Reprod. Dev.
48 339-343 (1997)).
It is known that ovulation in pigs o
DeSousa Paul Alexandre
King Timothy James
Wilmut Ian
Zhu Jie
Crouch Deborah
Earp David J.
Geron Corporation
Schiff J. Michael
Ton Thai-an N.
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