Intra-cytoplasmic sperm injection in a marsupial

Abstract

Here we report the first use of intra-cytoplasmic sperm injection (ICSI) in a marsupial, the tammar wallaby (Macropus eugenii ), to achieve in vitro fertilization and cleavage. A single epididymal spermatozoon was injected into the cytoplasm of each mature oocyte collected from Graafian follicles or from the oviduct within hours of ovulation. The day after sperm injection, oocytes were assessed for the presence of pronuclei and polar body extrusion and in vitro development was monitored for up to 4 days. After ICSI, three of four (75%) follicular and four of eight (50%) tubal oocytes underwent cleavage. The cleavage pattern was similar to that previously reported for in vivo fertilized oocytes placed in culture, where development also halted at the 4-to 8-cell stage. One-third of injected oocytes completed the second cleavage division, but only a single embryo reached the 8-cell stage. The success of ICSI in the tammar wallaby provided an opportunity to examine the influence of the mucoid coat that is deposited around oocytes passing through the oviduct after fertilization. The presence of a mucoid coat in tubal oocytes did not prevent fertilization by ICSI and the oocytes cleaved in vitro to a similar stage as follicular oocytes lacking a mucoid coat. Cell-zona and cell -cell adhesion occurred in embryos from follicular oocytes, suggesting that the mucoid coat is not essential for these processes. However, blastomeres were more closely apposed in embryos from tubal oocytes and cell-cell adhesion was more pronounced, indicating that the mucoid coat may be involved in maintaining the integrity of the conceptus during cleavage.

The production of mature oocytes from adult ovaries following primary follicle culture in a marsupial

“…Having achieved maturation to metaphase II, the next obvious step is to assess the developmental competence of oocytes by examining rates of fertilization and embryonic development. Unfortunately, the techniques required for such analysis are not yet established in marsupials (Mate 1996, Magarey & Mate 2003, Richings et al 2004. To date, successful IVF has only been achieved in M. domestica and ICSI in M. eugenii (Richings et al 2004), neither study, however, attempted surrogate transfer to follow embryonic development to birth.…”

WOMEN IN REPRODUCTIVE SCIENCE: Reproduction down under

“…With Geoff Shaw, Kath Handasyde and our students, we used these to reversetranslate this human contraceptive into an effective management tool for overabundant populations of kangaroos (Nave et al 2000(Nave et al , 2002 and koalas (Hynes et al 2011), now being used in the field for management of overabundant koalas by State authorities. On the flip side, my lab developed assisted reproductive techniques for marsupials, including the first in vitro fertilised marsupial embryo (Richings et al 2004), the first conceptus produced by artificial insemination (AI) (Paris et al 2005) and the first maturation of follicles in vitro after cold storage (Richings et al 2006). Injection through the shell coat that surrounds all marsupial conceptuses was challenging, as was attempting AI via the long median vagina of marsupials.…”

Artificial insemination in marsupials

“…As just discussed, female synchronization is a major problem for marsupials that needs to be addressed, but ICSI in marsupials (tammar wallaby and common wombat) by contrast appears relatively generic [101,105,106]. Sperm injection is now so well established for treatment of human infertility that several million babies have been produced and in some countries, it now is the preferred method of IVF.…”