Methods are provided for stimulating ovarian follicles in a mammal through activation of the mTor signaling pathway.

The present invention relates to a method of producing a non-human, mammalian oocyte carrying a modified target sequence in its genome, the method comprising the steps of introducing into a non-human, mammalian oocyte: (a) a clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein 9 (Cas9 protein) or a nucleic acid molecule encoding said Cas9 protein; and (b-i) a target sequence specific CRISPR RNA (crRNA) and a trans-activating crRNA (tracr RNA) or a nucleic acid molecule encoding said RNAs; or (b-ii) a chimaeric RNA sequence comprising a target sequence specific crRNA and tracrRNA or a nucleic acid molecule encoding said RNA; wherein the Cas9 protein introduced in (a) and the RNA sequence(s) introduced in (b-i) or (b-ii) form a protein/RNA complex that specifically binds to the target sequence and introduces a single or double strand break within the target sequence. The present invention further relates to the method of the invention, wherein the target sequence is modified by homologous recombination with a donor nucleic acid sequence further comprising the step: (c) introducing a nucleic acid molecule into the cell, wherein the nucleic acid molecule comprises the donor nucleic acid sequence and regions homologous to the target sequence. The present invention also relates to a method of producing a non-human mammal carrying a modified target sequence in its genome.

The present invention provides novel methods for improving the efficiency of artificial activation of unfertilized mammalian oocytes by reducing the intracellular concentration of Zn.sup.2+ in the oocyte. The methods of the invention may additionally comprise a preceding step of increasing the intracellular concentration of Ca.sup.2+ in the oocyte prior to reduction of the intracellular Zn.sup.2+ concentration. The invention further provides unfertilized oocytes activated by the disclosed methods and viable mammalian animals produced from unfertilized oocytes activated by the disclosed methods.

Methods of preparing ovarian tissue for primordial follicle growth are presented comprising the steps: providing an ovarian tissue sample comprising cortical tissue and stromal tissue; removing damaged tissue from the ovarian tissue sample where present; removing excess stromal tissue from the ovarian tissue sample where present; and then mechanically stretching the ovarian tissue sample along at least one dimension of the ovarian tissue sample, such that the size of the ovarian tissue sample along the at least one dimension is increased by at least 10%. Methods of growing viable oocyte in vitro, and methods of preparing individual ovarian follicles for growth are also presented.

Methods of producing human stem cells are disclosed for parthenogenetically activating human oocytes by manipulation of O.sub.2 tension, including manipulation of Ca.sup.2+ under high O.sub.2 tension and contacting oocytes with serine threonine kinase inhibitors under low O.sub.2 tension, isolating inner cell masses (ICMs) from the activated oocytes, and culturing the cells of the isolated ICMs under high O.sub.2 tension. Moreover, methods are described for the production of stems cells from activated oocytes in the absence of non-human animal products, including the use of human feeder cells/products for culturing ICM/stem cells. Stem cells produced by the disclosed methods are also described.

Methods and compositions for treating ovarian failure are provided. In one embodiment, the method includes administering stem cells into the ovary of a female subject in need of such treatment. The stem cells are preferably bone marrow derived stem cells (BMSC). In other embodiments, the stem cells are embryonic stem cells, adult stem cells, induced stem cells, induced pluripotent stem cells, umbilical cord blood cells, or combinations thereof. The stem cells can be autologous or heterologous. In one embodiment, the stem cells have the following surface marker profile: CD105 positive, CD166 positive, CD90 positive, and CD73 positive as well as CD14 negative, CD34 negative, CD45 negative, HLA-DR negative, and CD 19 negative. The stem cells are administered in an amount effective to restore ovarian hormone production and promote folliculogenesis.

The present invention provides a novel oocyte maturation medium or/and embryo culture medium with a chemically defined supplement to produce matured oocytes at high efficiency. The inventive medium or supplement comprises three growth factors, namely, fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF-1) in a synergistic combination. Methods for oocyte and embryo culture are also provided.

Use of Cistanche deserticola polysaccharide (CDP) in promoting the proliferation and differentiation of female germline stem cells (FGSCs) is provided. Specifically, the addition of CDP in an in vitro cultivation system can promote the proliferation and differentiation of FGSCs, and especially can enhance the in vitro directed differentiation of FGSCs into oocytes, which provides a new research reference for studying the generation of oocytes in vivo and in vitro and also brings a new hope for research on physiological infertility.

The disclosure relates to Bovine germplasm of Bos taurus HO840003210132823. Included in the present disclosure are cells comprising the genome of Bovine HO840003210132823 characterized by the presence of homozygous loci and spermatozoa obtained from said cells. Also provided by the present disclosure are tissue cultures of cells, animals obtained from said cells, and parts thereof, including F1 spermatozoa. The disclosure further provides for methods of breeding, selecting, and using the germplasm to improve existing commercial cattle herds generated from in vitro fertilization methods and progeny cattle obtained from in vitro fertilization and implantation and artificial insemination methods.

The invention relates to a carrying device for beverage cans which allows the manual carrying of beverage cans grouped together in the form of a “pack”, which device comprises a body devoid of side walls, having on its surface at least one opening defining a contour with proportions which allow the tight passage therethrough of a beverage can, the contour of said opening having a plurality of tabs that can be folded or bent in relation to the body itself and extending into the same opening, said tabs having a distribution according to at least one sequence, said sequence comprising two contiguous tabs followed by a gap followed by another tab followed by another gap.