Nitric oxide (NO) has emerged as one of several important intraovarian regulatory factors. In particular, NO has been implicated in the processes of ovulation and atresia-related apoptosis. The aim of the present study was to investigate the presence and distribution of the NO-generating nitric oxide synthase (NOS) enzymes in the ovary during follicular development, ovulation and luteal formation of the equine chorionic gonadotrophin (ECG)/human chorionic gonadotrophin (HCG)-primed rat. NADPH diaphorase activity was used as a histochemical marker for NOS within the ovary. Diaphorase reactivity was most abundant in the stroma (S) of the ovary and in the theca (T) layer of the follicle. In luteinized ovaries, weaker diaphorase reactivity was present within the corpora lutea (CL). Two different isoforms of NOS, the constitutively expressed endothelial NOS (eNOS) and the inducible isoform of NOS (iNOS), were immunolocalized in ovaries of immature rats and in ECG/HCG-primed rats during the periovulatory period from HCG injection until 2 days after ovulation. In addition, ovarian concentrations of eNOS and iNOS were quantified by immunoblotting. Immunoblotting with a monoclonal anti-eNOS antibody demonstrated the presence of eNOS mainly in the residual ovary (ROV) during the periovulatory period. In luteinized ovaries, higher concentrations of eNOS were seen in CL, while those in the ROV at this stage were lower than in the periovulatory ovary. Immature ovaries contained diminutive amounts of eNOS, detectable mostly in the ROV compartment. In contrast, iNOS was barely detectable during follicular development to the preovulatory stage. A slight elevation of iNOS was observed in the granulosa cells at 6 h after the HCG injection. The levels of iNOS during the luteal phase were also low. Immunohistochemical analysis using polyclonal eNOS and iNOS antibodies revealed the localization of these two isoforms primarily in the S and the T of the periovulatory ovary. In luteinized ovaries, positive immunoreactivity was also seen within the CL. With a monoclonal antibody against eNOS, intense immunoreactivity was observed in the S, T and within CL. There was a particularly strong staining in blood vessels. These data demonstrate the presence of an intraovarian NO-generating system. The localization of this system to the S, T and CL suggests a role for NO in the ovulatory process and in the regulation of CL function.
A method of heterotopic uterine transplantation was developed in the mouse as a model system for studies of uterine function and transplant immunology of the uterus. The model involved transplantation of the right uterine horn and the cervix by vascular anastomosis to a donor animal with the intact native uterus remaining in situ. F1-hybrids of inbred C57BL/6 x CBA/ca (B6 CBAF1) mice of 6-8 weeks of age (n=42) were used. The specific pelvic vascular anatomy of these mice was first examined by intra-aortal injection of a two-component silicon-rubber curing agent. The surgery of the donor animal involved microsurgical isolation of the right uterine horn and the cervix, with preserved vascular supply from the aorta through the right uterine artery. After isolation of the uterine horn with vascular supply and venous drainage, including approximately 3 mm of the inferior vena cava and aorta, the organ was put on ice. The recipient animal was prepared by exposing and mobilizing the infrarenal part of the aorta and the vena cava. The grafted uterus was placed in the abdomen on the left side and the aorta and vena cava of the graft were anastomosed end-to-side to the aorta and vena cava of the recipient animal with 11-0 sutures. The total time for these procedures declined with time and was 125+/-4 min for the last 28 operations. Viability of the uterus was confirmed, several days later, by demonstrating a blood flow similar to that of the native uterus, and histology of the grafted uterus demonstrated normal morphology, including intact ultrastructure of the endothelial cells. The overall survival rate of the recipient animals increased with learning from approximately 40% in animals 1-21 to 71% in animals 22-42. The proportion of viable grafts, as judged by normal blood flow and histology among the surviving mice was 25% in animals 1-21 and 87% in animals 22-42. An undisturbed function of the transplanted uterus horn was finally demonstrated by its ability to implant inserted blastocysts and to carry pregnancy with fetal weight being similar to that of fetuses in the native uterus and controls. In conclusion, this is the first report of successful transplantation of the uterus with proven functionality in the mouse. The model should be useful for many aspects of research in uterine physiology and pathophysiology.
Uterus transplantation may become the first available treatment for uterine factor infertility, which is due to the absence or malfunction of the uterus. Here we describe for the first time pregnancy after allogeneic uterus transplantation, as a proof of concept of uterine function in a transplanted uterus in a standardized animal model (rat) under immunosuppression.
Serum progesterone and human chorionic gonadotrophin (HCG) were analysed using a time-resolved fluoroimmunoassay in an unselected group made up of 158 women with clinical suspicions of abnormal early gestation. Only cases in which endovaginal sonography had failed to localize the pregnancy were included. A single HCG determination had no diagnostic value. On the other hand, a critical progesterone level of 30 nM was determined below which no viable intrauterine pregnancies were found. Eighty-eight per cent of the ectopic pregnancies (n = 97) and 83% of the spontaneous abortions had progesterone levels below this limit. The discriminatory efficacy of one single progesterone determination was independent of the actual HCG level and serial determinations of progesterone did not increase the discriminatory power.
In vitro perfusion may prove to be a suitable method to test viability and function of frozen-thawed whole ovaries contributing to the optimization of current cryopreservation protocols.
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