Changes in the activity of sperm nitric oxide synthase in the oviductal reservoir during ovulation

Abstract

Background:  The oviductal isthmus is known to act as a sperm reservoir in several mammalian species including mice, but it is still unclear how sperm are released from the reservoir after ovulation. Recently, nitric oxide (NO) was reported to have important roles as a mediator in various sperm functions, including hyperactivation and capacitation. Therefore, we have investigated the change of the activity of nitric oxide synthase (NOS) of sperm of the isthmus in relation to ovulation under in vivo fertilization conditions.

Methods and Results:  The sperm were collected from the isthmus and uterus of the female mated before or after ovulation. The NOS activity change was evaluated by using the β‐nicotinamide adenine dinucleotide phosphate‐diaphorase staining method, and sperm NOS activity was quantified by using NIH image software. The results showed that, in the reservoir, the peak intensity of sperm NOS activity was higher after ovulation (135.5 ± 22.4) than before ovulation (102.7 ± 15.5; P ≤ 0.05). After ovulation, the number of free sperm in the isthmus increased, and these sperm expressed strong NOS activity.

Conclusion:  The change of sperm NOS activity is related to their release from the epithelium of the oviductal reservoir. (Reprod Med Biol 2003; 2: 75–81)

INTRODUCTION

TO COMPLETE FERTILIZATION in the female reproductive tract, sperm must undergo a variety of the physiological events such as capacitation and hyperactivation until they reach the ampulla of the oviduct. ¹ , ² , ³ , ⁴ In several species of mammals including mice and humans, the distal portion of the isthmus of the oviduct is considered to act as a functional sperm reservoir. ⁵ , ⁶ The reservoir is formed by the interaction between the sperm head and the oviductal epithelial cells. It is postulated that the reservoir mainly serves three important roles: (i) prevention of polyspermic fertilization by allowing only a few sperm to reach the oocytes in the ampulla at an appropriate time; (ii) maintaining the fertility and viability of sperm between the onset of estrous and fertilization; and (iii) regulation of the processes of capacitation and motility hyperactivation within the reservoir. ⁷ , ⁸ When ovulation is initiated, the reserved sperm are gradually liberated from the interaction, and the released sperm subsequently fertilize the eggs. However, it is not well understood how sperm are released from the epithelial cells upon ovulation.

The activation of sperm motility (hyperactivation) is considered to be one of the factors involved in the release. ² , ⁸ In addition, hyperactivated motility is strongly related in vivo for getting sperm to the site of fertilization (ampulla), and to penetrate the cumulus matrix and zona pellucida. ⁹ , ¹⁰ Recently, nitric oxide (NO) is suggested to have important roles as a mediator of various sperm functions, including the motility. ¹¹ Nitric oxide is known to be a radical that plays important roles in the mechanisms of numerous physiological events including reproduction. ¹² Nitric oxide is a product of the conversion of l‐arginine to l‐citrulline via the action of the nitric oxide synthase (NOS), which requires reduced nicotinamide adenine dinucleotide phosphate (NADPH) as an essential cofactor. ¹³ , ¹⁴ Nitric oxide synthase has three isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Previous studies using antibodies against nNOS, eNOS, and iNOS have revealed that these isoforms are expressed in nearly all tissues, including the reproductive systems. ¹⁵ , ¹⁶ In mice and humans, NOS was localized in the head and tail regions of the sperm. ¹⁷ , ¹⁸ Moreover, NO production by sperm increases in a time‐dependent manner under capacitating conditions in vitro. ¹⁹

It is of interest whether NO production by sperm NOS is related to the release of the sperm from the oviductal epithelium before and after ovulation. To examine this, we used an NADPH‐diaphorase staining method with paraformaldehyde fixation, which can be used to detect NOS activity alone by abolishing the NADPH‐diaphorase activity not related to NOS. ²⁰ Here, we report that the NOS activity of sperm in the reservoir increased after ovulation.

MATERIALS AND METHODS

Animals

MATURE FEMALE AND male mice of the strain of the Institute of Cancer Research (ICR) were purchased from Japan SLC (Hamamatu, Japan). All procedures were performed in accordance with our institution's guidelines for the care and use of laboratory animals, and were approved by the Animal Research Committee of the Miyazaki Medical College.

Chemicals

All chemicals were purchased from Nacalai Tesque Inc. (Kyoto, Japan) unless stated otherwise.

Distribution of sperm in the lower isthmus of the mated females

Mature females were induced to ovulate and were mated at 3–4 h before their scheduled ovulation time. At 1–2 h after vaginal plug formation, the mated females were anesthetized with diethyl ether and subsequently perfused through the left ventricle with Bouin's fixative. The tissues of the ovary and uterus were dissected out en masse, and immersed in the same fixative overnight at room temperature. The specimens were dehydrated with ethanol and embedded in paraffin. Two micrometer thick sections were cut and stained with hematoxylin and eosin. The distribution of sperm in the isthmus region was determined by observation under a light microscope (BX‐50; Olympus, Tokyo, Japan).

Collection of sperm from mated females

Mature male (12–14 weeks old) and virgin female (8 week‐old) mice were used in the present study. All animals were maintained with the following lighting conditions: light, 15:00–05:00 hours; darkness, 05:00–15:00 hours. To synchronize the ovulation times of all female mice, the mice were injected intraperitoneally with 5 IU of pregnant mare serum gonadtrophin (PMSG) at 23:00 hours, and then with 5 IU of human chorionic gonadotropin (hCG) 48 h later. Ovulation in these animals occurred at approximately 12 h after the injection of hCG (11:00 hours). The time schedules of sperm collection from mated females are shown in Fig. 1. For the collection of sperm from females mated before ovulation, the estrous females were placed in the cage of a male at 07:00 hours, that is, 4 h before the scheduled ovulation time. Vaginal plug formation was checked at 08:00 hours. The females that formed vaginal plugs were killed by an overdose of anesthesia with diethyl ether at 09:00 hours, that is, 1–2 h after plug formation and approximately 2 h before ovulation. For the collection of sperm from females mated after ovulation, the females were placed in the cage of a male at 11:00 hours, that is, at the scheduled ovulation time, and the vaginal plug formation was checked at 12:00 hours. The females that formed vaginal plugs were killed at 13:00 hours, that is, 1–2 h after plug formation and approximately 2 h after ovulation. The uterus and lower isthmus were dissected out and immediately fixed with 4% paraformaldehyde in 0.1 mol/L phosphate buffer (PB), pH 7.4 for 1 h at 4°C. After fixation, the samples were minced, washed three times with 0.1 mol/L PB, and then checked for the presence of sperm. At the same time, the presence of ovulated eggs in the ampulla regions was confirmed.

Figure 1.

Diagram of the time schedules of sperm collection from mated females. hCG, human chorionic gonadotropin; OV, scheduled ovulation time; PMSG, pregnant mare serum gonadotrophin; SC, sperm collection; #, check of plug formation; dark area: period of copulation.

Nicotinamide adenine dinucleotide phosphate‐diaphorase staining

Nicotinamide adenine dinucleotide phosphate‐diaphorase staining was carried out by using the method of Scherer‐Singler et al. ²¹ Briefly, the samples were incubated with a freshly prepared solution of β‐NADPH (1 mg/mL; Sigma, St Louis, MO, USA), nitroblue tetrazolium (0.2 mg/mL; Sigma) and Triton X‐100 (5.0 µL/mL; Sigma) in 0.1 mol/L PB, pH 7.4, at 37°C for 3 h. For controls, the sperm of all samples were incubated with staining solution without β‐NADPH. After NADPH‐diaphorase staining, the samples were washed three times with 0.1 mol/L PB at 4°C, air‐dried onto silane‐coated glass slides, and observed with a light microscope (LM: Axioplan; Zeiss, Oberkochen, Germany). The changes of solutions during the process of staining were conducted meticulously to prevent accidental staining errors.

Analysis of the nicotinamide adenine dinucleotide phosphate‐diaphorase activity

Analysis of the NADPH‐diaphorase activity was performed as follows. Bright‐field microscopic images of the sperm of each sample were recorded by using the AxioVision 3.0 program (Zeiss), which was connected to a LM via a digital camera system (Axiocam; Zeiss). The images were converted from a color to a gray‐scale by using Photoshop 4.0 software (Adobe Systems, San Jose, CA, USA). By using NIH image 1.62 software (National Institute of Health, Bethesda, MD, USA), the indicated stained region shown in Fig. 2 with the Line Selection Tool was selected, and the staining intensity by using the analyze‐measure function was measured. The maximum gray values within the selected region were then displayed in the information window. The maximum gray values were defined as the peak intensity of NADPH‐diaphorase staining of the sperm. As the intensity of the staining showed almost the same value in every region of the midpiece in each spermatozoon, the intensity, by focusing on the middle part of the midpiece as a representative value, was measured.

Figure 2.

Method of measurement of the level of sperm nitric oxide synthase (NOS) activity. The activity of NOS was found only in midpiece. The stained region was chosen perpendicularly to the long axis of the sperm midpiece, and the maximum value of the reaction color intensity was recorded as the measured value.

Relationship between the state and nitric oxide synthase activity of the sperm in the isthmus before and after ovulation

Each sperm (a total of 100 sperm per sample) collected from the isthmus before and after ovulation was determined in terms of the state and the NOS activity. The states of the sperm were categorized into two types as follows: (i) the sperm associating with the oviductal epithelium‐like cell cluster (associating sperm); and (ii) the free sperm.

Statistical analysis

To compare the NOS activity of the sperm from the uterus and isthmus, 50 sperm per sample, both before and after ovulation, were randomly measured and analyzed. To determine the relationship between the state and the NOS activity of the sperm in the isthmus, more than 50 sperm (a total of 100 sperm) per sample before and after ovulation were measured and analyzed. For each sample, the means of the peak value ± SEM were computed. For each experimental group, three animals were used. Statistical analysis was performed by using the Student's t‐test. Differences between the values were judged significant at P < 0.05.

RESULTS

Distribution of sperm in the lower isthmus of the mated females

IN THE MATED females before ovulation, sperm were observed in the crypts of the lower isthmus, and the sperm heads were associated with the epithelial cells of the crypt (Fig. 3).

Figure 3.

Light microscopic image of the oviductal reservoir (isthmus) of a mated female. Hematoxylin and eosin staining. Many sperm heads (arrows) are found in the crypts of the oviductal isthmus. Bar = 10 µm.

Nicotinamide adenine dinucleotide phosphate‐diaphorase activity of the sperm in the reproductive tract

In this experiment, sperm were collected from mated females 2 h before or after the scheduled ovulation time (1–2 h after plug formation). Ovulated eggs were found only in the oviductal ampullae collected at 2 h after the scheduled ovulation time. Regardless of the site and the time of sperm collection, NADPH‐diaphorase activity was detected only in the midpiece of the sperm. No reaction was found in the sperm head or principal piece of the sperm tail (Fig. 4). In the females, 2 h after ovulation, the level of NADPH‐diaphorase activity of sperm in the lower isthmus was significantly stronger than that of sperm in the uterus. In the females at 2 h before ovulation, the level of NADPH‐diaphorase activity of sperm in the isthmus was almost the same as that of sperm in the uterus.

Figure 4.

Light microscopic image of sperm from the uterus (a,c) or isthmus (b,d) of mated females. β‐Nicotinamide adenine dinucleotide phosphate (NADPH) and nitroblue tetrazolium staining. Sperm from the (a) uterus and (b) isthmus of a female mated before ovulation, and sperm from the (c) uterus of a female mated after ovulation. All these sperm were stained in the same pattern, with a weak reaction confined to the midpiece, and no reaction is found in the sperm head or principal piece of the tail. By contrast, sperm from the (d) isthmus of a female mated after ovulation have a strong reaction for NADPH‐diaphorase activity only in the midpiece of the tail. Bar = 5 µm.

To quantify the level of enzyme activity, the value of the peak staining intensity was measured by using the intensity‐measuring function of the NIH image; the relationship between the peak value and number of the sperm is shown in histograms (Fig. 5). At this time, the value of the background level in sperm heads, the principal piece of the tail and the sperm‐free region, was 35–45 values on average. Moreover, for control, the midpiece of sperm treated with the staining solution without β‐NADPH was also 35–45 values on average. More than 75% of sperm, both from the uterus 2 h before or after ovulation and the isthmus 2 h before ovulation, showed low values in the range of 80–120 (Fig. 5a–c). In contrast, more than 70% of the isthmus sperm 3 h after ovulation showed a high value in the range of 120–160 (Fig. 5d). The mean values ± SEM of the intensity for the experimental groups were as follows: the sperm from the uterus (99.0 ± 10.8) and isthmus (102.7 ± 15.5) at 2 h before ovulation, and sperm from the uterus (98.6 ± 10.2) and isthmus (135.5 ± 22.4) at 2 h after ovulation. Among these groups, the difference of the peak value between sperm from the isthmus at 2 h after ovulation and sperm of the other three groups was significant (P ≤ 0.05).

Figure 5.

Histograms of the staining intensity of nitric oxide synthase (NOS) activity in individual sperm in four different conditions; sperm from the (a) uterus and (b) isthmus of females mated before ovulation, and sperm from the (c) uterus and (d) isthmus of females mated after ovulation. The peak intensity of the reaction of the uterus sperm did not change (a) before and (c) after ovulation. By contrast, the peak intensity of the reaction of the isthmus sperm shifted from low (b; before ovulation) to high (d; after ovulation).

Relationship between the state and nitric oxide synthase activity of the sperm in the isthmus before and after ovulation

The standard of NOS activity was set up on the basis of the result that compared the value of reaction intensity of the isthmus and uterus sperm before or after ovulation as follows: the intensity of 120 values or less was low activity and the intensity of over 120 values was high activity. The relationship between the state and the NOS activity of sperm in the isthmus before and after ovulation is shown in Table 1. The number of sperm with a high activity of NOS after ovulation significantly increased as compared with that of before ovulation in both associating sperm and free sperm (P ≤ 0.05). The number of free sperm after ovulation (49.6% ± 6.4) increased as compared with that of before ovulation (32% ± 3.6; P ≤ 0.05).

Table 1.

Relationship between the state and nitric oxide synthase (NOS) activity of the sperm in the isthmus before and after ovulation

	Total no. sperm	Mean percentage ± SD
		Associating sperm†		Free sperm‡
		–	+	–	+
Before ovulation	300 (n = 3)	58.3 ± 6.5a	9.7 ± 3.1c	19.7 ± 2.1e	12.3 ± 2.1g
After ovulation	300 (n = 3)	11.0 ± 2.6b	39.3 ± 3.8d	12.0 ± 2.0f	37.7% ± 4.7h

Experiments were conducted three times. †Sperm associating with the oviductal epithelium‐like cell cluster; ‡sperm not associating with the oviductal epithelium‐like cell cluster; (–) low value of NOS activity (<120); (+) high value of NOS activity (>120). P < 0.05: a versus b; c versus d; e versus f; g versus h.

DISCUSSION

IN THE PRESENT study, we investigated sperm NOS activity by using a NADPH‐diaphorase staining method to clarify the roles of NO produced by the sperm distributed in the isthmus of mated female mice (Fig. 3). The results showed that the NOS activity of the sperm collected from the isthmus of mated females after ovulation remarkably increased (4, 5). In addition, the result of the relationship between the state and the NOS activity of the sperm in the isthmus showed that the sperm associated with the epithelium had a lower activity of NOS until the onset of ovulation, whereas after ovulation, these sperm expressed a higher activity of NOS and became free from the epithelial cells. These findings suggest that the sperm release from the interaction with the epithelium is related to the change of sperm NOS activity in the isthmus after ovulation. In the physiological conditions in vitro, it is demonstrated that the quantity of NO produced by sperm themselves increase during capacitation in vitro in humans and mice. ¹⁸ , ^{19 , 22} Moreover, sperm motility was reduced by using NOS inhibitors such as N^G‐nitro‐l‐arginine methyl ester (l‐NAME), nitro‐l‐arginine, and methyl‐l‐arginine in mice, humans and hamsters. ¹⁸ , ^{22 , 23} By contrast, it is demonstrated that sperm motility was decreased by a high concentration of sodium nitroprusside, which is an NO‐releasing compound. ²⁴ , ²⁵ , ²⁶ These reports suggest that endogenous NO produced by sperm themselves is involved in the facilitation of sperm activation, and that the concentration of NO must be regulated closely to be beneficial to sperm.

Sperm in the lower isthmus before ovulation are significantly less motile, but most of the sperm after ovulation has the characteristic flagellar bending pattern of the hyperactivated motility in the mouse and rabbit. ⁵ , ²⁷ , ²⁸ , ²⁹ Moreover, sperm hyperactivation facilitates the release of sperm from the oviductal reservoir. ⁹ In the previous in vitro studies, the interaction between the sperm and the oviductal cells presumably maintained the sperm fertility and/or motility in cattle and humans. ³⁰ , ³¹ , ³² Taking these reports and our present study results into consideration, we hypothesize that the sperm, which enter the oviductal reservoir, remain inactivated, owing to the suppression of NOS activity until the onset of ovulation, whereas after ovulation, the suppression of sperm NOS activity is overcome and facilitates the release of sperm from the oviductal epithelium for fertilization.

It is still unclear the mechanism by which sperm NOS is activated before and after ovulation in the present study. The activation of constitutive NOS of the sperm is stimulated by a Ca²⁺ ionophore (A23187), ³³ and the Ca²⁺ uptake of sperm bound to epithelial cells is suppressed in the oviduct. ³⁴ However, little is known about the biochemical pathways regulating Ca²⁺ uptake of the sperm during ovulation. In several species of mammals such as hamsters, horses, and cattle, co‐incubation studies of sperm with oviductal epithelial cells in vitro showed that carbohydrates mediate the sperm interaction with the oviductal epithelial cells in the sperm reservoir. ³⁵ , ³⁶ , ³⁷ , ³⁸ Combined use of the quantitative method of sperm NOS activity determination used in the present study and other in vitro methods may provide further clues about the mechanism of the release of sperm from the reservoir.

In addition, in the present study, NOS activity was not detected in the head region of the sperm in which NOS was localized. A possible reason is as follows: because the quantity of NO, which the sperm itself produce, is as low as 1.2–2.9 µmol/L per 10⁶ viable sperm, the NOS activity of the sperm head was out of the range of detection when the β‐NADPH staining method was used in the present study.

In conclusion, the sperm NOS activity in the oviductal reservoir changed during ovulation. Our results suggest that NO produced by the sperm in the isthmus is related to sperm activation, and facilitates the release of sperm from the reservoir. Thus, the activity of sperm NOS is thought to be one of the factors for the mechanism of sperm release from the reservoir. In addition, this is the first report on sperm NOS activity change in the oviductal reservoir before and after ovulation in vivo in mice to be conducted.