concluded that this model may be useful to better understand pre-fertilization sperm maturation events in the possum, which could facilitate the development of IVF technology. q 2000 Elsevier
Science B.V. All rights reserved.
Keywords: Possum; Sperm transport; Superovulation; Artificial insemination
1. Introduction
Timed observations on the fate of spermatozoa after their deposition in the female Ž
tract have been made in only five marsupial species, Macropus eugenii Tyndale-Biscoe .
Ž and Rodger, 1978 , Antechinus stuartii Selwood, 1982; Taggart and Temple-Smith,
. Ž
. Ž
1991 , Sminthopsis crassicaudata Breed et al., 1989 , Didelphis Õirginiana Rodger .
Ž and Bedford, 1982a,b; Bedford et al., 1984 and A. agilis Shimmin et al., 1999;
. Taggart et al., 1999 . Semen is deposited in the upper part of the urogenital sinus in
Ž .
marsupials and progresses up the lateral vaginae to the twin cervices Rodger, 1991 . From the cervices, sperm move through the utero–tubal junction into the lower isthmus
region of the oviduct. The utero–tubal junction forms a barrier to sperm migration up Ž
. the female tract in eutherian mammals Hunter, 1988 . In marsupials, however, this
region appears to present little, if any, barrier to sperm progression into the isthmus of Ž
. Ž
. the oviduct Taggart, 1994 . Both dasyurids Taggart and Temple-Smith, 1991 and
Ž .
didelphids Bedford et al., 1984 transport sperm efficiently with 1 in 20 ejaculated spermatozoa reaching the isthmus of the oviduct. In these species, sperm are stored in
Ž .
specialised isthmic crypts for up to 2 weeks located near the utero–tubal junction Ž
. reviewed by Taggart, 1994 . Similar isthmic crypts have not been previously reported
Ž .
Ž in the brushtail possum Arnold and Shorey, 1985 or macropodid marsupials Tyndale-
. Biscoe and Rodger, 1978 .
In addition to its role in sperm storage, the isthmus is thought to have an important Ž
. role in the capacitation of spermatozoa in eutherian mammals Yanagimachi, 1994 .
Capacitation is an obligatory maturational event which sperm must undergo in the female tract before achieving full fertilizing potential. It generally occurs in the lower
segment of the oviduct, and is currently thought to involve the removal of surface
Ž coating materials and an increased permeability to metal ions Sidhu and Guraya, 1989;
. Yanagimachi, 1994 .
Although this is yet to be definitively established, it appears that marsupial spermato- zoa also undergo a period of capacitation, but exactly what regions of the female
Ž .
reproductive tract are involved in this process is unknown Mate and Rodger, 1996 . Further, the mechanisms involved appear quite different, not only from eutherians but
also between Australian and American marsupials. In American species, separation of Ž
paired spermatozoa is highly correlated with the acquisition of fertilizing ability Moore .
and Taggart, 1993 , while in several Australian marsupials, reorientation of the head Ž
. region of highly motile spermatozoa to a thumbtack T-shape configuration is thought
Ž .
to be an indicator of capacitation Bedford and Breed, 1994; Molinia et al., 1998 . The study of sperm transport in the possum depends quite critically on being able to
accurately and consistently manipulate their breeding in captivity. The unreliability of
possum natural mating behaviour has led to the development of a laparoscopic artificial Ž
. Ž
. insemination AI procedure Molinia et al., 1998 . As possums are monovular, it has
been necessary to concurrently develop superovulation and AI protocols to maximise potential numbers of ovulated eggs available for fertilization in vivo. Further, hormone
manipulation permits control of the timing of insemination relative to ovulation, and fertilization. Using both AI and superovulation with the exogenous hormones pregnant
Ž .
Ž .
mare serum gonadotrophin PMSG rLH, fertilized eggs or embryos 1–2 per possum Ž
. are consistently recovered Molinia et al., 1998 . Accordingly, the combination of
superovulation and AI provides a model system to investigate sperm transport and pre-fertilization events in the possum.
The main aim of the current study was to investigate sperm transport in vivo following intrauterine AI of PMSGrLH superovulated possums. Specific objectives
were to determine the distribution of spermatozoa through the female reproductive tract in relation to ovulation, to characterise the epithelial lining of the oviduct with particular
emphasis on the presence of any sperm storage regions within the lower isthmus, and to examine the morphology and motility of spermatozoa within each of the isthmus, middle
and ampulla oviduct segments.
2. Materials and methods
