Mating evokes a characteristic pattern of molecular and cellular events in the rodent reproductive tract, including an infiltration of the endometrial stroma and uterine lumen with activated macrophages and granulocytes, which closely resembles a classic inflammatory response. Previous studies in mice indicate that these cellular changes are associated with, and are largely a consequence of, an upregulated synthesis and release of granulocyte-macrophage colony-stimulating factor (GM-CSF) from the uterine epithelium in response to seminal fluid. The aim of this study was to investigate further the origin and nature of the factors present in seminal fluid that trigger the GM-CSF response. It was found that the characteristic increase in uterine expression of mRNA encoding GM-CSF and release of GM-CSF bioactivity from uterine epithelial cells into the luminal cavity seen after mating with intact or vasectomized males was no longer evident in matings with male mice from whom the seminal vesicles had been surgically removed. The extent of inflammatory leucocyte infiltration into the endometrium was also reduced; the most notable effect was a complete absence of the exocytosis of neutrophils into the luminal cavity normally seen after matings with intact or vasectomized males. Bioassay of the GM-CSF output of oestrous endometrial cells after culture with crude or Sephacryl S-400 chromatographed fractions of seminal vesicle fluid showed that the GM-CSF stimulating activity was predominantly associated with protein moieties in seminal vesicle fluid of approximately 650,000 M(r) and 100,000-400,000 M(r). These data confirm the presence in seminal vesicle fluid of specific factors that initiate an inflammatory response in the uterus after mating through upregulating GM-CSF synthesis in the uterine epithelium. The significance of the cytokine release and cellular changes induced by seminal plasma for implantation of the conceptus and pregnancy outcome remain to be determined.

The rat testis contains a large population of resident macrophages, the physiological roles of which are yet to be established. To investigate the functional capacity of these cells, we have analyzed the secretion of the cytokines interleukin (IL)-1, IL-6, tumor necrosis factor alpha (TNF alpha), and granulocyte macrophage-colony stimulating factor (GM-CSF) by isolated testicular macrophages (TMs) and, for comparison, by isolated rat peritoneal macrophages (PMs). Cells were cultured for 48 h in serum-free medium alone or with lipopolysaccharide (LPS, 10 micrograms/ml) and/or recombinant interferon-gamma (rIFN gamma, 200 U/ml). Specific bioassays were used to measure cytokines in the media collected from cultures. Basal production of IL-1, TNF alpha, and IL-6 by TMs and PMs were similar, but TMs produced 8-fold greater levels of GM-CSF than did PMs. LPS, alone or in combination with IFN gamma, significantly enhanced the secretion of all cytokines by PMs (340-840% increase). LPS alone had little effect on TM secretion except to reduce GM-CSF levels some 4-fold. The addition of LPS and IFN gamma increased IL-1, IL-6, and TNF alpha levels (200-750% increase) and reduced GM-CSF levels to 45% of basal levels. Treatment of cultures with indomethacin to minimize prostaglandin production enhanced the LPS-induced effects in both cell types. Expression of the mRNA for each cytokine in cultures of testicular and peritoneal macrophages, as well as in intact testis, was confirmed by reverse transcription polymerase chain reaction. These studies indicate that macrophages resident within the rat testis have a novel cytokine secretion profile and an altered responsiveness to inflammatory activators compared with macrophages from the peritoneal cavity. This may be important in physiological processes in the testis and may contribute to the dysfunctional afferent immune activity thought to underlie the immunologically privileged status of the testes.

Our studies suggest that semen may play a critical role in providing the antigenic and environmental signals necessary to initiate an appropriate maternal immune response to the conceptus during pregnancy.

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The rhythmic expression of clock and clock-controlled genes in the rat oviduct was investigated by real time RT-PCR. per1, per2, Clock, Bmal1, cry1 and cry2 were all expressed in the oviduct. With 4-hourly sampling over 24 h in a normal photoperiod, analysis of variance indicated that per2 and Bmal1 had highly significant sinusoidal-like changes with an amplitude of 3- and 10-fold respectively. Of the other clock genes, per1 and cry1 had non-significant rhythm amplitudes of 2.5- and 1.8-fold respectively. Using the same experimental approach the rhythmic expression of Bmal1, per1 and per2 mRNA in the liver was found to be highly significant with amplitudes of approximately 20-, 10- and 5-fold respectively. The expression of the clock-controlled transcription factors DBP and Rev-erb alpha showed significant rhythmicity in the oviduct with 5-fold changes in amplitude for both genes. Plasminogen activator inhibitor-1 (PAI-1), which has been implicated in oviduct function during the preimplantation period, also had a significant rhythm of expression (2.5-fold amplitude), peaking at the same time as the other clock-controlled genes, DBP and Rev-erb alpha. These results show for the first time that the female reproductive tract is inherently rhythmic and suggests that the developing embryo may be subjected to rhythmic changes in the environment created by the oviduct during transition to the uterus.