Binding of porcine spermatozoa to uterine epithelial cells modulates the female immune response and might indicate the formation of a pre-oviductal sperm reservoir.

Abstract

Inseminations in pigs are characterized by the tremendous amount of spermatozoa needed for successful fertilisation. lf, in contrast, spermatozoa are deposited at the tip of the uterine horn a fraction of the usual porcine insemination dose suffices (Johnson 1991, Vazquez et al. 2005). It thus seems to be the uterine passage where the need for such high sperm numbers arises. In the past the provision of sufficient sperm numbers to reach acceptable fertility rates did not pose a problem due to the abundance of spermatozoa in one single boar ejaculate. However, modern biotechnological procedures, such as sex sorting of spermatozoa, require insemination of sperm portions containing no more then 50 x 106spermatozoa. To facilitate insemination with such small sperm doses also for conventional Al-techniques, more knowledge has to be gathered about fundamental sperm transport and selection mechanisms within the uterus. Previous studies on the pig uterus (Lovell & Getty 1968), the utero-tubal junction (Rodriguez-Martinez et al. 1990) and the oviduct (Wagner et al. 2002) suggested that spermatozoa are indeed subject to close interaction and even binding with the epithelial structures of the female genital tract. The present study aimed to further our understanding of such interactions specifically in the porcine uterus. For this purpose an ex vivo model was developed using uterine segments of 10 cm derived from 50 freshly slaughtered peri-ovulatory German Landrace gilts. In each segment 100 x 106 spermatozoa were incubated for 60 min at 38°C. The sperm cells originated from ejaculates provided by 4 boars of the same breed and of proven fertility. Previous to incubation spermatozoa were either washed and diluted in the semen extender Androhep or diluted with autologous seminal plasma without further washing. The spermatozoa were subsequently flushed out of the segments, counted and their viability parameters were established flow cytometrically using the stains PI and JC1 for membrane integrity and mitochondrial membrane potential respectively. The results indicated a retention of viable spermatozoa within the uterine cavity since only 55 ± 7% of the intact spermatozoa (PI-4C1+) were rediscovered in the flushing, while the damaged sperm population (PI+ /JC1-) was flushed out almost in its entity (93 ± 12%; p < 0.03). The effect was more emphasised in the sperm population, which had been washed and diluted in Androhep' (p < 0.05). The location of the uterine segments in relation to the cervix had no effect on the numbers of recovered sperm cells. Neither were differences observed between segments from the right and left uterine horn. In order to determine the physiological reasons behind the observed uterine sperm retention an in vivo experiment was performed looking for changes in uterine gene expression in response to insemination by measuring endometrial mRNA concentration. Because many processes concerning reproduction such as ovulation and implantation involve the immune system, the