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a Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri 63110
b Department of Physiology, Southern Illinois University at Carbondale School of Medicine, Carbondale, Illinois 62901
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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NO is synthesized from L-arginine by nitric oxide synthase (NOS) isozymes, yielding NO and L-citrulline [11]. To date, three genes encoding NOSs with different biochemical characteristics have been isolated [12, 13]. Two constitutively expressed isoforms were first identified in the endothelium (eNOS) and brain [11, 12]. An inducible isoform (iNOS) has been found in many cells and is correlated with cytostatic and cytotoxic events [13].
Studies in our laboratory and others have demonstrated that eNOS and iNOS are expressed in a cell- and development-specific manner in the superovulated rodent ovary [4, 14]. Furthermore, both isoforms were regulated by gonadotropins [14]. Ovarian eNOS and iNOS also have been detected during the estrous cycle in rats [15, 16]. NO is synthesized by rat [2, 10] and human [7, 17] ovarian cells, and treatment of rats with pharmacological NOS inhibitors lowered the number of ovulated oocytes both in perfused ovaries and in vivo [1, 3, 5, 18].
The importance of independent roles of iNOS and eNOS in female reproduction has been studied in our laboratory using mice deficient in iNOS (iNOS-knockout) or eNOS (eNOS-knockout). Both iNOS-knockout and eNOS-knockout female mice showed a significant reduction in ovulation rate relative to wild-type (WT) controls [6, 19]. In normal breeding we have observed a lower pregnancy rate in eNOS-knockout females compared with either WT or iNOS-knockout females. Furthermore, among those mice that become pregnant, the number of pups born per litter was fewer, and these pups had a high initial mortality rate [6].
The objective of the present study was to examine the influence of genotype on several aspects of the normal reproductive capacity, namely, cyclicity, ovulation rate, steroidogenesis, and gonadotropin levels during the estrous cycle in WT, iNOS-, and eNOS-deficient mice.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Wild-type (129Sv/Ev) females were originally obtained from Taconic Laboratories (Germantown, NY). Inducible NOS-knockout mice on a 129Sv/Ev/C57BL6 genetic background were originally obtained from Dr. John Mudgett, Merck Research Laboratories, Rahway, NJ [20], and their offspring were bred as homozygote matings (iNOS-/-). Endothelial NOS-knockout mice on a 129Sv/Ev/C57BL6 genetic background were originally obtained from Drs. Paul Huang and Mark Fishman, Harvard Medical School, Boston, MA [21], and their offspring were bred either as homozygote matings (eNOS-/-) or heterozygote females (eNOS+/-) with eNOS-knockout males [6]. Pups were weaned at 21 days of age, at which time all pups were sexed. Pups from heterozygote matings were ear notched for identification purposes, and a 1-cm segment of the tail was removed and prepared for isolation of genomic DNA. The genotype of the offspring was determined by Southern blot analysis [6]. Mice were maintained in a barrier facility with a 12L:12D cycle (lights-on from 0600 h to 1800 h) so that midnight (2400 h) was the midpoint of the dark cycle. Temperature was maintained at 25°C, and mice were provided food and water ad libitum.
The animal protocol used was approved by the Washington University Medical School institutional committee on laboratory animal care and was conducted in accordance with the NIH guide for the care and use of laboratory animals.
Classification of the Estrous Cycle and Tissue Collection
To examine the influence of genotype on the length of the estrous cycle, vaginal smears from WT, iNOS-knockout, and eNOS-knockout female mice at 13–16 wk of age were monitored daily between 1400 h and 1500 h. The mouse's tail was raised and a cotton swab, prepared from tooth picks and sterile cotton, was inserted into the vagina and rotated. When the swab was removed, it was rolled onto a microscope slide. The smears were classified into one of four phases of estrus: a thin smear of leukocytes and elongated nucleated epithelium indicated proestrus; large cornified epithelial cells were exclusively found in estrus; metestrus was marked by a thick smear composed of equal numbers of nucleated epithelial cells and leukocytes; and a smear consisting almost exclusively of leukocytes depicted diestrus [22, 23]. Each cycle length was determined as the length of time between two consecutive occurrences of estrus. After the mice had progressed through at least three consecutive estrous cycles, the length of the estrous cycle and the number of days spent at each stage of the cycle were evaluated.
To determine whether iNOS and eNOS deficiency affected ovarian weight, ovarian morphology, and hormone concentrations, mice were weighed and killed at 1500 h and 1830 h on proestrus and at 1500 h on the first day of diestrus. Blood samples were collected by cardiac puncture, and plasma from these samples was stored at -20°C. Given the limitations of the small amount of plasma obtained from a limited number of mice, we chose to examine the levels of E2 and LH at proestrus, and E2 and progesterone (P4) at diestrus of the estrous cycle. Visual examination of uteri provided additional confirmation of the phase of the estrous cycle. Animals in late proestrus had large uteri with a substantial amount of intraluminal fluid. Mice in estrus had smaller uteri and a small amount of intraluminal fluid. In turn, small uteri without intraluminal fluid were characteristic of mice in diestrus [16]. All animals were killed by CO2 asphyxiation, followed by cervical dislocation.
Ovulation Efficiency
To determine the number of ovulated oocytes during the estrous cycle, WT, iNOS-knockout, and eNOS-knockout females (n = 4 mice per genotype) were killed at 0800 h in the morning of estrus. For each mouse, the enlarged and translucent ampulla of each oviduct was excised and trimmed, and the ovulated oocyte-cumulus complexes were recovered into a Petri dish with Dulbecco's PBS (Sigma Chemical Co., St. Louis, MO). Oocytes were counted and data are expressed as the mean number of ovulated oocytes per mouse per genotype [6].
Hormone Assays
E2 and P4 concentrations were determined in plasma using commercial RIA kits (Abbott Laboratories, Abbott Park, IL, for E2; Diagnostic Products Corp., Los Angeles, CA, for P4). Samples were analyzed singly or in duplicate when sufficient plasma was available. The inter- and intraassay coefficients of variation were 4.5% and 4.1% for E2, and 5.1% and 2.6% for P4, respectively. Plasma LH concentration was determined in a double-antibody RIA using established procedures [24]. Hormone standards and reagents were supplied by the National Hormone and Pituitary Program (Bethesda, MD). Goat anti-rabbit 
-globulin (Calbiochem, La Jolla, CA) was used as second antibody. LH results were expressed in term of the NIADDK-rat reference preparation RP-3 (Rockville, MD). The sensitivity of the LH assay was 0.2 ng/ml and the intraassay coefficient of variation was 6.8%.
Ovarian Histology
To determine whether lack of NO synthesis influences ovarian development, ovaries collected on proestrus and diestrus of the estrous cycle (n = 4 mice per genotype) were examined. Both ovaries were removed, cleaned of extraneous tissue, weighed, and fixed in Bouin's solution [25]. After 20-h fixation, the tissue was dehydrated in a graded ethanol series and embedded in paraffin wax. Serial sections (6 µm thick) of ovaries (4 ovaries per stage of the cycle per genotype) were stained with hematoxylin and eosin [22]. For each ovary (average 200 sections per ovary), every fifth section was examined to obtain an overall view of the follicular populations at proestrus and diestrus of the estrous cycle in WT, iNOS-knockout, and eNOS-knockout mice. Sexually mature females contain a heterogenous population of follicles at different developmental stages [26, 27]; therefore, the specific analysis was limited to large antral preovulatory follicles in proestrous mice and small antral follicles and corpora lutea (CL) in diestrous mice. At least two sets of CL are present in diestrous ovaries. The most recent CL (newly formed) stain blue with hematoxylin; the latter (older) are stained more heavily with eosin [22, 28].
Statistical Analysis
All data are presented as the mean ± SEM. Data on the length of the estrous cycle, hormone concentrations, and ovulation efficiency were analyzed using ANOVA, and multiple comparisons were made with Bonferroni's t-test. Number of days spent in each stage of the estrus cycle was analyzed via the chi-square test [29].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine whether iNOS-knockout and eNOS-knockout females were undergoing normal estrous cycling, vaginal smears were taken daily, examined histologically, and compared with those of WT females. Control WT females reached estrus every 4 to 5 days (Fig. 1), and their smear patterns at each stage displayed a characteristic morphology. The length of the estrous cycle was not significantly altered (p = 0.06) in iNOS-knockout mice compared with control WT mice (Fig. 1). However, we consistently observed a longer diestrus in iNOS-knockout mice compared with WT mice, indicating a moderate alteration in cyclicity. In contrast, eNOS-knockout females showed significantly lengthened estrous cycles, reaching estrus every 6.6 ± 0.6 days, relative to the cycles of control WT females (p < 0.03) (Fig. 1). The estrous cycle of eNOS-knockout females included a significantly extended diestrus (p < 0.03), whereas the duration of all other stages was not significantly changed compared to those of WT mice (Fig. 2).
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Effects of iNOS and eNOS Deficiency on Ovarian Weight and Steroidogenesis
We observed no effect of genotype on body weight (overall mean for WT, iNOS-knockout, and eNOS-knockout females = 24.0 ± 0.5 g) and ovarian weight (overall mean = 5.4 ± 0.2 mg) during the estrous cycle (Table 1). Furthermore, there was no influence of iNOS or eNOS deficiency on the ratio of ovarian weight to body weight compared with that for control WT females (Table 1).
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Plasma concentrations of E2, LH, and P4 in WT, iNOS-knockout, and eNOS-knockout females are shown in Table 2. In WT females, the highest level of plasma E2 was observed at 1500 h on the afternoon of proestrus. By 1830 h that same day, E2 had declined significantly (p < 0.001) and remained low at diestrus (Table 2). Inducible NOS-knockout females also showed cyclic changes in plasma E2 levels, but the pattern was different from that in WT females (Table 2). At 1500 h on proestrus, the level of E2 was significantly lower than observed in WT females. The E2 level then increased and reached a peak at 1830 h of proestrus followed by a decline at diestrus (p < 0.05). In eNOS-knockout females the preovulatory peak of E2 occurred at 1830 h of proestrus, similar to the time in iNOS-knockout mice; however, the concentration of E2 was 5-fold and 3-fold higher (p < 0.05) than the levels observed in WT and iNOS-knockout mice, respectively (Table 2). The E2 level of eNOS-knockout mice decreased at diestrus but remained higher (2-fold; p < 0.05) than the E2 concentrations for WT and iNOS-knockout mice.
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We observed no effect of genotype on the plasma LH level at 1500 h of proestrus (Table 2). There was a significant rise in LH concentration at 1830 h of proestrus for WT (14-fold, p < 0.05) and eNOS-knockout (3-fold; p < 0.05) mice, but no significant difference between genotypes. A 2-fold increase in LH levels at 1830 h of proestrus in iNOS-knockout females did not achieve significance (p = 0.06) (Table 2).
There was no difference in plasma P4 levels between WT and iNOS-knockout mice on the first day of diestrus. However, eNOS-knockout mice showed a significantly higher (3-fold; p < 0.05) plasma P4 level on the first day of diestrus relative to either control WT or iNOS-knockout females (Table 2).
Ovulation Rate and Ovarian Morphology of WT, iNOS-Knockout, and eNOS-Knockout Mice
There was no influence of iNOS deficiency on ovulation rate compared with that in WT females (7.50 ± 0.86 oocytes per mouse and 8.25 ± 1.03 oocytes per mouse, respectively) (Fig. 3). However, the ovulatory efficiency of eNOS-knockout females was significantly reduced (4.75 ± 1.10 oocytes per mouse) relative to that in control WT or iNOS-knockout females (inhibition of 42% and 36%, respectively; p < 0.05) (Fig. 3).
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To assess the effect of disruption of the iNOS or eNOS gene on ovarian morphology, histological studies were conducted on 4 ovaries at each stage of the cycle for each genotype. The ovaries obtained from sexually mature cycling mice contain a heterologous population of follicles at different stages of development, among which primary and secondary follicles, various sizes of antral follicles, and CL could be observed. Paraffin-embedded sections of ovaries from iNOS-knockout and eNOS-knockout females at proestrus and diestrus of the estrous cycle were compared with those of the WT females (Fig. 4). The characteristic antral preovulatory follicle with its oocyte surrounded by a well-formed cumulus was indicative of late proestrus. The ovary of diestrus was marked by an abundance of healthy primary and secondary follicles. The number of large antral follicles in iNOS-knockout mice was not different from that in the WT mice (4.3 ± 1.2 vs. 4.7 ± 0.3 per ovary) (Fig. 4, A and C, respectively). Ovaries from eNOS-knockout females collected during proestrus contained fewer large antral follicles (2.3 ± 0.5 per ovary; p < 0.05) compared with ovaries from WT or iNOS-knockout mice (Fig. 4E). Similar to the findings for proestrous ovaries, no morphological differences were noted between diestrous ovaries collected from WT and iNOS-knockout mice (Fig. 4, B and D, respectively). Small antral follicles and newly formed CL stained blue with hematoxylin, as well as older red-stained (with eosin) CL, were observed in ovaries from both WT and iNOS-knockout females (Fig. 4, B and D, respectively). Ovaries from eNOS-knockout mice during diestrus showed fewer newly formed CL, whose number was comparable to the number of ovulated oocytes. Some ovaries from eNOS-knockout females also contained luteinized follicles and many growing antral follicles undergoing atresia (Fig. 4F).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition to abnormal cyclicity, our studies on ovarian morphology have shown that eNOS deficiency affects follicular development [31]. Ovaries from eNOS-deficient females obtained during proestrus contained a lower number of large antral follicles and fewer CL at diestrus compared with ovaries from WT or iNOS-deficient females. Supporting a role for eNOS-derived NO in follicular development is the positive staining for eNOS in granulosa and thecal cells in the follicles [4, 6, 14] and the oocyte-specific expression of eNOS both within the follicle and after ovulation [6]. Furthermore, recent studies in patients undergoing in vitro fertilization treatment demonstrated that circulating nitrite/nitrate levels increase with follicular development and are correlated with follicular size [17]. Thus, the relationship between the NO pathway and follicular growth could be necessary for both follicular and oocyte development.
There is now compelling evidence that NO also plays a role in the ovulatory process. It has been reported that inhibition of NOS with competitive inhibitors resulted in fewer ovulations both in vivo and with use of perfusion systems in rats and rabbits [1, 3, 14, 18, 32]. A recent study in our laboratory demonstrated that superovulated eNOS-deficient female mice showed a significant reduction in the number of ovulated oocytes relative to WT females [6]. Furthermore, ovulated oocytes obtained from eNOS-knockout females showed abnormalities in meiotic maturation and increased oocyte cell death relative to oocytes obtained from WT females, suggesting possible defects in follicular and oocyte development [6]. In the present study we observed that ovulatory efficiency of eNOS-knockout females was also significantly reduced during the estrous cycle relative to that of WT and iNOS-knockout females. We observed no effect of iNOS deficiency on the ovulation rate during the estrous cycle compared with that in WT females. Although many investigators have suggested a role for iNOS in the ovulatory process, the data are variable. For example, the pattern of ovarian iNOS expression is not consistent between laboratories [4, 7, 14]. Inducible NOS protein was localized exclusively in the thecal cell layers and stroma of preovulatory follicles in rat ovaries, and its level is regulated by gonadotropins, suggesting that iNOS/NO may function as a signal for somatic cells to properly modulate the theca and tunica albuginea of the follicle necessary for the rupture of the follicular wall [4, 14]. However, in contrast to iNOS protein levels, iNOS mRNA was detected only in granulosa cells of secondary preantral follicles with the highest amounts in unstimulated rat ovaries [7]. In another study performed in rats, a significant increase in iNOS gene expression was shown in the early stage of first proestrus, followed by a decline in its transcription in the late stage of proestrus [16]. Interestingly, the same investigators reported that the significant increase in iNOS protein expression in the late stage of first proestrus, just prior to first ovulation, coincided with elevated levels of serum E2 and LH [16]. Due to the variable data in the literature, it is difficult to make firm conclusions concerning the function and regulation of ovarian iNOS during the periovulatory period. Our data, however, support a primary role for eNOS rather than iNOS-derived NO in ovulation.
The extension of the estrous cycle, along with the reduced number of developing follicles and ovulated oocytes in eNOS-knockout mice, suggested potential regulation of the neuroendocrine-ovarian axis by NO and/or local control of ovarian steroidogenesis by NO. In terms of regulation of neuroendocrine function, constitutive NOSs have been localized in several hypothalamic areas [33] and have also been shown in the posterior [34] and anterior pituitary [35]. Recent in vivo and in vitro investigations have demonstrated that NO is involved in the generation of both pulsatile and surge patterns of LH secretion through the regulation of GnRH [36–39], and therefore may influence the production of ovarian steroids via this mechanism. Furthermore, because NO is also involved in the control of vascular tone, it may indirectly influence hormone release by regulating hypophyseal portal blood flow. In the present study we observed a 14-fold rise in LH levels at 1830 h compared with 1500 h during proestrus in WT females. In both eNOS-knockout and iNOS-knockout mice, peak LH levels were lower than that observed in WT mice. The LH peak for eNOS-deficient females was only 3-fold higher at 1830 h relative to its level at 1500 h, and was significantly lower compared with that of WT females at 1830 h. While LH levels did rise in iNOS-knockout mice between 1500 h and 1830 h, the change was not statistically significant. The inability to detect a significant change in peak LH levels at 1830 h versus 1500 h in iNOS-knockout mice suggests that the LH peak may have occurred earlier, since we observed no dysfunction in the ovulation rate or level of ovarian steroids of iNOS-knockout females compared with WT females. A close estimate of the LH value and time of the true peak could be determined only by more frequent sampling of plasma in individual iNOS- and eNOS-deficient mice, since the time of LH rise is not entirely synchronous among animals [40, 41]. Furthermore, the maximal serum LH concentration on the afternoon of proestrus in rats varies greatly from animal to animal [41].
An alternative explanation for the dysregulation of the estrous cycle of eNOS-knockout mice may relate to ovarian steroid production. It is now well established that the surge of gonadotropins leading to ovulation is regulated by E2 and P4 [42–46]. E2 is the primary trigger for the preovulatory gonadotropin surge, and in rats its serum levels normally fall rapidly on proestrus afternoon during the LH surge [43, 47]. Although E2 is essential for the gonadotropin surge, Turgeon [47] demonstrated that its continued secretion during proestrus in rats is not necessary for the surge and is, in fact, inhibitory to the surge. Thus, abnormal elevations in E2 levels may result in inhibition of the LH surge. Several studies have demonstrated that NO negatively regulates E2 synthesis in human and rat luteal cells by binding to aromatase and directly inhibiting its activity [1, 7, 8, 48]. Therefore, a lack of ovarian NO would result in increased E2 levels. The results obtained in the present study show that plasma E2 levels of eNOS-deficient females at late proestrus and diestrus were significantly higher than those of WT or iNOS-deficient females. We have also observed higher E2 levels in superovulated eNOS-knockout mice compared with WT mice just after ovulation [6]. These data support a role for eNOS-derived NO in the local regulation of E2 synthesis. In addition to increased E2 levels, we observed elevated levels of P4 in eNOS-knockout mice during diestrus. It is well established that plasma P4 drops in the morning of diestrus in mice when regression of the CL of the cycle normally occurs [46]. The extended diestrus in eNOS-deficient mice, in combination with elevated E2 and P4 secretion during diestrus, suggests that eNOS/NO may locally regulate steroidogenesis and modulate the life span of CL.
An extended luteal life span could also be the result of increased plasma prolactin levels. Indeed, transgenic mice and rats overexpressing high levels of human growth hormone (hGH), which binds murine prolactin receptors [49, 50], show significant prolongation of the estrous cycle and elevated P4 levels similarly to eNOS-knockout mice. However, in contrast to the subfertile eNOS-knockout mice, transgenic mice expressing high levels of hGH [49] are fertile and show increased ovulation rate relative to that in control mice. A lack of NO may also result in altered prolactin levels either due to direct regulation of prolactin secretion by NO or via an indirect effect of NO on E2 synthesis. These data suggest that some of the reproductive defects observed in eNOS-knockout mice may be due to abnormal prolactin secretion.
Taken together, the deficiency of eNOS-derived NO in eNOS-knockout females, correlated with dysfunction in cyclicity, ovarian morphology, and alteration in steroidogenesis, strongly supports the concept that eNOS/NO has important roles in ovulation and follicular development. Furthermore, the iNOS- and eNOS-deficient mice provide excellent tools to separate the function for each isoform and to study the roles of iNOS and eNOS in ovarian processes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Current address: Loyola University, Stritch School of Medicine, Department of Obstetrics and Gynecology, 2160 S. First Avenue, Maywood, IL 60153.
Accepted: February 24, 1999.
Received: September 10, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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