Process for Enriching a Population of Sperm Cells

• US 20080039680A1
• Filed: 07/22/2005
• Published: 02/14/2008
• Est. Priority Date: 07/22/2004
• Status: Active Grant

• Current Assignee: Inguran LLC
• Original Assignee: Monsanto Technology LLC (Bayer AG)
• Inventors: Graham, Jeffrey
• Granted Patent: US 7,833,147 B2
• Time in Patent Office: Days
• US Class Current: 600/35
• CPC Class Codes: A61K 35/52 Sperm; Prostate; Seminal fluid; Leydig cells of testes
• CPC Class Codes: C12N 5/0612 sorting of gametes, e.g. according to sex or motility
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 1/42 Low-temperature sample treatment, e.g. cryofixation
• CPC Class Codes: G01N 15/1433 using image recognition

Abstract

Processes for selectively enriching a population of viable sperm cells with respect to a characteristic without physically sorting the cells are disclosed. The cells contained in such an enriched population benefit from the advantage of not being subjected to a sorting process. Processes of inseminating a female mammal and processes of forming a sperm dispersion utilizing the processes of selectively enriching a population of viable sperm cells are also disclosed.

Claims

1. A process for selectively decreasing the capacity of a subpopulation of sperm cells in a sperm cell dispersion to fertilize an egg, the process comprising:

forming a dispersion of labeled sperm cells in a liquid, the liquid comprising a chemical agent or having a temperature that induces sperm immotility, wherein the presence, absence or amount of the label associated with a sperm cell indicates a genetic, proteomic, structural, or functional characteristic of a subpopulation of sperm cells in the dispersion;

optically inspecting the dispersion to identify individual sperm cells as members of the subpopulation;

determining the position of members of the subpopulation in the dispersion; and

delivering a dose of energy to different positions within the dispersion to selectively decrease the capacity of members of the subpopulation to fertilize an egg without similarly affecting sperm cells at other positions in the dispersion.

5. The process of any of claims 1-4, wherein the liquid comprises a chemical agent that induces sperm immotility.

6. The process of any of claims 1-4, wherein the liquid has a temperature that induces sperm immotility.

7. The process of any of claims 1-4, wherein the liquid comprises a chemical agent and has a temperature that induces sperm immotility.

8. The process of any of claims 1-7, wherein the amount of the label associated with the sperm cell indicates that the sperm cell is an X chromosome-bearing sperm cell.

9. The process of any of claims 1-7, wherein the amount of the label associated with the sperm cell indicates that the sperm cell is a Y chromosome-bearing sperm cell.

10. The process of any of claims 1-9, wherein the label is selected from the group consisting of fluorescent dyes, DNA selective dyes, polyamides, oligonucleotides, and a polypeptide that binds to a surface specific characteristic of a sperm cell.

11. The process of claim 10, wherein the label is a DNA selective fluorescent dye.

12. The process of claim 11, wherein the label is Hoechst 33342, Hoechst 33258, or SYBR-14.

13. The process of any of claims 1-7, wherein the dose of energy is selected from the group consisting of radiation beams, laser beams, collimated non-laser light, focused non-laser light, and focused ultrasonic energy.

14. The process of any of claims 1-7, wherein the process further comprises purifying the sperm cells not receiving a dose of energy, the non-dosed cells.

15. The process of claim 14, wherein purifying the non-dosed cells comprises centrifuging the dispersion and removing the dosed cells.

16. The process of claim 14, wherein purifying the non-dosed cells comprises contacting the dispersion with a high-density medium.

17. The process of any of claims 1-12, wherein optically inspecting the dispersion to identify individual sperm cells as members of the subpopulation comprises optically inspecting a captured image of the cells.

18. The process of any of claims 1-12, wherein prior to optically inspecting the dispersion, the dispersion is distributed onto a multi-well plate.

19. The process of claim 18, wherein the multi-well plate is a 96 or 384 well plate.

20. The process of any of claims 1-12, wherein the dose of energy is sufficient to decrease the viability of the members of the subpopulation as compared to the viability of sperm cells not receiving a dose of energy.

21. The process of any of claims 1-12, wherein the dose of energy is sufficient to cause the death of the members of the subpopulation.

22. The process of any of claims 1-6 wherein the process further comprises cryopreserving the dispersion subsequent to delivering the dose of energy.

24. The process of claim 2 or any of claims 5-14 when dependent upon claim 2, wherein the insemination of the female mammal occurs within about 7 days, 5 days, 3 days, 2 days, or 1 day after the dosing of the cells is complete.

25. The process of claim 3 or any of claims 5-14 when dependent upon claim 3, wherein the fertilization of an egg, in vitro, occurs within about 7 days, 5 days, 3 days, 2 days, or 1 day after the dosing of the cells is complete.

26. The process of claim 3 or any of claims 5-14 when dependent upon claim 3, wherein the process further comprises introducing the fertilized egg into the uterus of a female mammal.

27. The process of claim 2 or any of claims 5-14 when dependent upon claim 2, wherein the dispersion is not cryopreserved prior to insemination of a female mammal.

28. The process of claim 2 or any of claims 5-14 when dependent upon claim 2, wherein the dispersion is cryopreserved prior to insemination of a female mammal.

29. The process of claim 3 or any of claims 5-14 when dependent upon claim 3, wherein the enriched dispersion is not cryopreserved prior to fertilization of an egg.

30. The process of claim 3 or any of claims 5-14 when dependent upon claim 3, wherein the enriched dispersion is cryopreserved prior to fertilization of an egg.

40. The process of any of claims 1-14 and 39, the process further comprising:

labeling the dosed dispersion with an additional label, wherein the presence, absence or amount of the additional label associated with a sperm cell indicates a genetic, proteomic, structural, or functional characteristic of a subpopulation of sperm cells in the dosed dispersion;

optically inspecting the dosed dispersion to identify individual sperm cells as members of the subpopulation;

determining the position of members of the subpopulation in the dosed dispersion; and

delivering a dose of energy to different positions within the dispersion to selectively decrease the capacity of members of the subpopulation to fertilize an egg without similarly affecting sperm cells at other positions in the dispersion.

41. The process of any of claims 1-14, wherein the temperature of the labeled sperm cells is about 0°

C. to about 15°

C.

42. The process of any of claims 1-14, wherein the temperature of the labeled sperm cells is about 1°

C. to about 10°

C.

43. The process of any of claims 1-14, wherein the temperature of the labeled sperm cells is about 2°

C. to about 8°

C.

44. The process of any of claims 1-14, wherein the temperature of the labeled sperm cells is about 3°

C. to about 6°

C.

45. The process of any of claims 1-14, wherein the temperature of the labeled sperm cells is about 4°

C. to about 5°

C.

46. The process of any of claims 1-14, wherein the temperature of the labeled sperm cells is about 5°

C.

47. The process of any of claims 1-14, wherein the temperature of the labeled sperm cells is about 4°

C.

2. A process for inseminating a female mammal with an enriched sperm cell population, the process comprising:

forming a dispersion of labeled sperm cells in a liquid, the liquid comprising a chemical agent or having a temperature that induces sperm immotility wherein the presence, absence or amount of the label associated with a sperm cell indicates a genetic, proteomic structural, or functional characteristic of a subpopulation of sperm cells in the dispersion;

optically inspecting the dispersion to identify individual sperm cells as members of the subpopulation;

determining the position of members of the subpopulation in the dispersion;

delivering a dose of energy to different positions within the dispersion to selectively decrease the capacity of members of the subpopulation to fertilize an egg without similarly affecting sperm cells at other positions in the dispersion, and

thereafter inseminating a female mammal with the dispersion or a derivative thereof.

23. The process of any of claims 2,3,5, 6, or 7 wherein the female mammal is a bovine, equine, or porcine.

3. A process for in vitro fertilization, the process comprising:

forming a dispersion of labeled sperm cells in a liquid, the liquid comprising a chemical agent or having a temperature that induces sperm immotility wherein the presence, absence or amount of the label associated with a sperm cell indicates a genetic, proteomic structural, or functional characteristic of a subpopulation of sperm cells in the dispersion;

optically inspecting the dispersion to identify individual sperm cells as members of the subpopulation;

determining the position of members of the subpopulation in the dispersion;

delivering a dose of energy to different positions within the dispersion to selectively decrease the capacity of members of the subpopulation to fertilize an egg without similarly affecting sperm cells at other positions in the dispersion; and

thereafter fertilizing an egg, in vitro, using the dispersion or a derivative thereof.

4. A process for forming a frozen sperm dispersion, the process comprising:

forming a dispersion of labeled sperm cells in a liquid, the liquid comprising a chemical agent or having a temperature that induces sperm immotility wherein the presence, absence or amount of the label associated with a sperm cell indicates a genetic, proteomic structural, or functional characteristic of a subpopulation of sperm cells in the dispersion;

optically inspecting the dispersion to identify individual sperm cells as members of the subpopulation;

determining the position of members of the subpopulation in the dispersion;

delivering a dose of energy to different positions within the dispersion to selectively decrease the capacity of members of the subpopulation to fertilize an egg without similarly affecting sperm cells at other positions in the dispersion;

and, thereafter cryopreserving the dispersion.

31. The process of any of claims 4, 5, 6, or 7 wherein the sperm cells not receiving a dose of energy comprise at least 85% X chromosome bearing sperm cells.

32. The process of any of claims 4, 5, 6, or 7 wherein the sperm cells not receiving a dose of energy comprise at least 85% Y chromosome bearing sperm cells.

33. The process of claim 4, 5, 6, or 7 wherein the sperm cells not receiving a dose of energy comprise at least 90% X chromosome bearing sperm cells.

34. The process of claim 4, 5, 6, or 7 wherein the sperm cells not receiving a dose of energy comprise at least 90% Y chromosome bearing sperm cells.

35. The process of claim 4, 5, 6, or 7, wherein the sperm cells not receiving a dose of energy comprise at least 95% X chromosome bearing sperm cells.

36. The process of claim 4, 5, 6, or 7, wherein the sperm cells not receiving a dose of energy comprise at least 95% Y chromosome bearing sperm cells.

37. The process of claim 4, 5, 6, or 7, wherein the sperm cells not receiving a dose of energy comprise at least 97% X chromosome bearing sperm cells.

38. The process of claim 4, 5, 6, or 7, wherein the sperm cells not receiving a dose of energy comprise at least 97% Y chromosome bearing sperm cells.

39. The method of claim 4 or any of claims 31-38, wherein the dosed dispersion is placed into a straw before freezing.