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Abstract

Nitric oxide produced from l-arginine by nitric oxide synthase (NOS) acts in a variety of biological processes via the stimulation of guanylyl cyclase and subsequent elevation of cGMP. Constitutive, calcium-dependent isoforms of NOS are found in endothelial cells (eNOS) and neurones (nNOS), while macrophages express an inducible, calcium-independent isoform (iNOS) in response to the action of certain cytokines or bacterial endotoxin. While the regulation of NOS by exogenous glucocorticoids and steroid hormones is well documented, the effects of endogenous steroid hormones on NOS activity, such as those released during the oestrous cycle, is unknown. Here we demonstrate, using specific antibodies for eNOS, nNOS and iNOS, the presence of NOS in the epithelium of rat fallopian tubes at pro-oestrus, late pro-oestrus, oestrus, metoestrus and dioestrus. Western blot analysis of rat fallopian tube homogenates revealed a protein band at approximately 125 kDa which was recognised by antibodies to different isoforms of NOS, but no bands at the expected molecular weights (eNOS, 140 kDa; nNOS, 160 kDa; iNOS, 135 kDa). NOS activity in fallopian tubes was measured by the conversion of l-[³H]arginine to l-[³H]citrulline. Both calcium-dependent and -independent NOS activities were present. However, in late pro-oestrus when circulating oestrogens are low, NOS activity was reduced in comparison to all other stages of the oestrous cycle. Thus we show that NOS is present in the epithelial lining of the fallopian tube and is recognised at a previously undescribed molecular weight. The changes in NOS activity in these cells during the oestrous cycle may modulate tube motility and contribute to sucessful fertility.

Journal of Endocrinology (1995) 146, 149–157

Abstract

We studied the expression of epidermal growth factor (EGF) receptor protein and messenger RNA (mRNA) in human fallopian tubes at three stages of the menstrual cycle: early follicular (n=3), late follicular (n=3) and luteal (n=3). Immunohistochemical studies in the ampullary portion of the tubes showed that specific staining was localized to the epithelium and the vascular endothelium. Staining of the epithelium was intense at the late follicular and luteal stages, while it was weak at the early follicular stage. ¹²⁵I-EGF binding study in the tubal plasma membranes revealed a class of high-affinity EGF receptors. Although dissociation constants were similar between the stages, numbers of binding sites at the late follicular and luteal stages were significantly (^P<0·01) greater than those at the early follicular stage. Western blotting showed that tubal plasma membranes contain M _r 170 000 EGF receptor protein. The amounts were significantly (P<0·01, n=3) greater at the late follicular and luteal stages than those at the early follicular stage. Reverse transcription and polymerase chain reaction (RT-PCR) revealed that EGF receptor mRNA was expressed in all the 9 RNA samples (n=3 for each stage) from the tubal ampullary portion. The amounts were significantly (P<0·01, n=3) greater at the late follicular and luteal stages than those at the early follicular stage (by a competitive PCR). Increase in the amounts of EGF receptor protein and mRNA occurred in association with an increase in serum oestradiol but not progesterone levels. Next we examined whether EGF receptor and its ligands (EGF and transforming growth factor a) are directly induced by oestrogen. We found that specific staining for EGF receptor and its ligands in the tubal epithelium was detected (by immunohistochemistry) in postmenopausal women with oestrogen replacement (n = 3), but not in subjects without oestrogen replacement (n=3). These results suggested that EGF receptors in the human tubal epithelium are expressed in relation to specific stages of the menstrual cycle and that the expression may be induced by oestrogen.

Journal of Endocrinology (1995) 147, 553–563

SUMMARY

A study was made of the effect of ligating the Fallopian tube or uterine horn near the tubouterine junction at specific time intervals after mating (½–6 hr) on fertilization in superovulated rabbits. A total of 140 does were used and altogether 2630 eggs were studied. Observations were made on the does' response to gonadotrophin treatment; the rate of egg development; the occurrence of abnormal eggs; the number of spermatozoa in fertilized eggs; the transfer of fertilized eggs to recipient does and the length of the female genital tract. Rabbit semen was collected by means of an improved type of artificial vagina.

The main results following ligation were as follows: when one Fallopian tube was ligated at the time intervals stated in parentheses, the mean proportion of eggs fertilized was 1·6% (1¼ hr), 8·8% (1¾ hr), 12·9% (2 hr), 16·1% (2¼ hr), 29·8% (2½ hr), 59·5% (2¾ hr), 85·9% (3 hr), 38·4% (3¼ hr), 73·7% (3½ hr), 50% (4 hr), 96·4% (5 hr) and 100% (6 hr). Out of a total of 1006 eggs from non-ligated (control) Fallopian tubes, 95·5% were fertilized. Following ligation of one uterine horn 30 min, 1 hr or 2 hr after mating, 9·1, 38·7 and 55·2%, respectively, of the eggs were fertilized.

It is concluded that although some spermatozoa may reach the tubo-uterine junction soon after mating, 2–5 hr are required before the number of spermatozoa entering the Fallopian tube is sufficiently high to produce maximum fertilization.

Incorporation of [U-¹⁴C]glucose in vitro was determined in ampullary, ampullary–isthmic junction and isthmic segments of the Fallopian tube and uterus of intact (oestrous) rabbits and also at various times after mating. In the oestrous animals, incorporation was slightly higher in the ampullary segment compared with the isthmic and the ampullary–isthmic junction and finally the uterus. The pattern changed significantly in the pregnant animals. At 14 and 24 h post coitum, the pattern of incorporation was similar to that found at oestrus except for an increase in uterine incorporation. However, at 48 and 72 h post coitum, incorporation by the isthmic segment and the uterus was significantly increased compared with corresponding values at oestrus, and 14 and 24 h post coitum.

The implication of these changes during egg transport through the Fallopian tube is discussed.

SUMMARY

At and for about 3 days after oestrus the utero-tubal junction of the ewe has a valve-like action which prevents fluid under pressure in the uterine (Fallopian) tube from entering the uterus.

This action appears to be the result of oestrogen-induced oedema and flexure of the wall of the utero-tubal junction.

SUMMARY

Ram spermatozoa were found at the ovarian end of the uterine (Fallopian) tubes of ewes at about 3 hr after mating. No radiopaque medium passed from the vagina to the uterine horns of rabbits following vulval stimulation and copulation. Ram spermatozoa entered the tubes of ewes through a valve-like utero-tubal junction which prevented fluid from passing in the opposite direction. Almost all ram spermatozoa became immotile within 8 hr of their introduction into the tubes of ewes. The method of sperm transport in the female genital tract is discussed.

SUMMARY

Using the techniques of sperm transfer into the oviduct of recipient females, or ovum transfer to the site of capacitation, it has been shown that the capacitating activity of the female genital tract may be modified by oestrogen and by progesterone. The normal capacitating potential of the Fallopian tube is diminished after ovariectomy, and is restored by oestrogen injection. In the absence of oestrogen (in the ovariectomized and adrenalectomized female) this activity is still maintained at a basal level sufficient to capacitate the numbers of sperm which normally enter the Fallopian tube after coitus. Capacitation is not significantly disturbed in the Fallopian tube of the intact progesterone-dominated female (Chang, 1958), and the innate basal capacitating activity persists in the Fallopian tubes of ovariectomized females treated with progesterone.

The ability of the uterus to bring about complete capacitation is lost in the progesterone-dominated female. Sperm are completely capacitated in about 11 hr. in the oestrous uterus, but only partial capacitation occurred during this period in the uterus of the ovariectomized rabbit. As judged by the time of penetration of eggs placed into the uterus at different times after insemination, sperm in the ovariectomized uterus did not acquire the capacity to fertilize for approximately 20 hr. Injection of up to 1000 i.u. human chorionic gonadotrophin at the time of insemination did not result in significant depression of capacitation in the uterus.

It is concluded that the rabbit oviduct has a greater innate potential for capacitation than the uterus, though this potential is increased in both sites by oestrogen stimulation. In contrast to its depressive effect on capacitation in the uterus, progesterone apparently does not significantly affect capacitation in the oviduct, even in the absence of the ovaries. This implies that modification of the endocrine status of the female rabbit would not produce a total contraceptive block of capacitation per se.

SUMMARY

The technique of tracing the movement of 'artificial eggs' (radioactive spheres) by autoradiography was used to study the effects of progesterone and oestrogen either alone, or in combination, on egg movement through the reproductive tract of ovariectomized rabbits.

Ovariectomy was performed 7 days before the transport experiments, except in one treatment group when it was done 35 days previously. The animals were autopsied at 8, 24, 48 or 56 hr. after insertion of the spheres into the ampullae of the Fallopian tubes. There were five or more animals in each group except one.

The effects of various hormonal replacements were studied. It was found that removal of the ovaries, whether 7 or 35 days before the transport experiments commenced, caused extremely irregular movement of the spheres through the Fallopian tube. Marked differences were recorded between the behaviour of the spheres in animals ovariectomized for the shorter as compared with the longer period.

None of the hormonal treatments produced normal movement of the spheres through the Fallopian tube. Progesterone delayed movement of the spheres through the ampulla very markedly, but if the spheres reached the isthmus they passed into the uterus on all occasions. Neither the isthmus nor the utero-tubal junction in animals so treated proved a barrier to the movement of the spheres into the uterus. Movement of the spheres through the uterus of these animals was inhibited, and most of the spheres recovered were found in the half of the uterus nearest to the utero-tubal junction.

Oestradiol benzoate, in the dose administered, had an accelerating effect on the passage of the spheres through the tube, and also through the uterus. Some spheres were found to have entered the vagina in three out of the four time-groups studied. It was observed that despite the accelerating effect of oestrogen on the movement of the spheres through the Fallopian tube a certain percentage, even after the longest interval studied, was trapped at the junction of the ampulla and the isthmus.

The two groups treated with oestrogen and progesterone in combination tended to produce results similar to those observed after treatment with either progesterone or oestrogen alone, depending on which hormone was dominant.

It is concluded that the amount of oestrogen present is critical in ensuring rapid transport through the ampulla, but that a proper balance of oestrogen and progesterone must be maintained to ensure retention of the spheres or eggs in the isthmus until the correct time for entry into the uterus. It is clear that this pattern of egg movement through the Fallopian tube cannot be re-created by fixed daily doses of progesterone and oestrogen.