"Light" epithelial cells of swine and bovine oviducts

The ultrastructure of oviduct epithelium of clinically healthy cows and 15 sows was investigated using scanning and transmission electron microscopy. In all parts of the oviduct, ciliated and non-ciliated epithelial cells are present, but their number varies in both the investigated animals in different regions of the oviduct, depending on the phase of the estrous cycle. In addition to ciliated cells with numerous cilia on their luminal surface, so-called pale ciliary cells were found in all parts of the oviduct of cows and sows. The cytoplasm of these cells is electron-lucent, their luminal surface carries few cilia and short microvilli. The apical cytoplasm contains species specific secretory granules, which means that these cells have features characteristic of both secretory and ciliated cells. It is suggested that the pale ciliated and non-ciliated secretory cells are functional stages of the same tubar epithelium cell, and that the transformation between these two cell types is regulated by functional requirements of the organ in different phases of the estrous cycle.     

Ultrastructural changes in the mammary glands of white rats during the estrus cycle

Secretory granules of milk protein were found in the apical cytoplasm of some ductal cells in the mammary gland of non-lactating albino rats. During the estrous period, the diestrous phase, the number of "coated" vesicles increased essentially. Some of the "coated" vesicles contained secretory granules and granular debris. The functional relationship of the "coated" vesicles to lysosomes and to some other cytoplasmic structures is discussed. The results suggest that during the estrous phase formation and release of secretory products prevail, whereas at the diestrous the process of their removal and destruction is predominant.     

Steroid receptors in the developing and the adult rabbit endocervix and in endocervical epithelial cells isolated by flow cytometry

Immunocytochemistry with monoclonal antibodies H222 and JZB39 was used to study nuclear estrogen (ER) and progesterone (PgR) receptors, respectively, in the cervix during differentiation and in the adult rabbit. The undifferentiated state of the cervix of 2-week-old rabbits correlates with a paucity of immunoreactive nuclear ER, while the epithelium of most of these animals showed moderate immunostaining for the nuclear PgR. The cervical epithelium, stroma and muscle cells of 1-month-old rabbits, showed weak immunostaining for the ER, while staining for PgR remained comparable to that of 2-week-old rabbits. For 2-4-month old rabbits the epithelium was characterized by moderate immunostaining for the nuclear ER and strong immunostaining for the PgR. Strong, heterogeneous immunostaining for nuclear ER and PgR receptors in endocervical epithelial cells from 6-month-old (adult), estrous rabbits suggested there are subpopulations of cells that express differential sensitivity to steroid hormones. In order to characterize such subpopulations, live endocervical epithelial cells were sorted with a flow cytometer on the basis of forward angle light scatter (FSC) and side scatter (SSC) signals which correlated with cell size and secretory granule content, respectively. Secretory cells, as verified by ultrastructural analysis and histochemical staining, expressed the highest FSC and SSC signals and were designated fraction "a". Changes in the hormonal status of the animals altered the intrinsic light scatter properties of fraction "a" cells as follows: maximum FSC and SSC signals were reported for cells from estrous animals; ovariectomy or progesterone-dominance decreased cell size (FCS) and secretory granule content (SSC), while treatment of ovariectomized rabbits with estradiol increased both parameters. When fraction "a" cells from estrous rabbits were incubated with the monoclonal antibodies, two distinct subpopulations of secretory cells were identified by intensity and pattern of nuclear staining for the ER and PgR. Changes in the hormonal status of the animals produced changes in the intensity of nuclear immunostaining, however both cell types remained distinguishable on the basis of immunostain pattern reflecting either permanent or transitory differences in them, and differential hormone sensitivity. The presence of nuclear ER and PgR proteins in these cells confirms their function is bireceptor-mediated.     

Light and electron microscopic observation on the cervical epithelium of the rabbit. I

The light and electron microscopy of the cervical epithelium of ovulatory, estrous, and long-term ovariectomized rabbits have been studied to determine what structural changes occur under different hormonal conditions. The percentage of nonciliated secretory cells is 49.6 in ovulatory, 43.6 in estrous, and 23.7 in long-term ovariectomized rabbits, and of ciliated cells is 50.2 in ovulatory, 56.2 in estrous, and 76.3 in long-term ovariectomized animals. The values for the ovulatory and estrous rabbits are significantly different at the P less than 0.05 level from those of the ovariectomized animals. In all 3 groups the general ultrastructure of the normal ciliated cells is similar. Interestingly, the Golgi complex is very prominent in all. Glycogen bodies occur frequently only in ciliated cells of ovariectomized and occasionally of estrous animals. Abnormalities in ciliation are quite common in the ovariectomized rabbits. The structure of the nonciliated secretory cells varies appreciably within and between the 3 groups. In these cells from well-developed epithelia of certain ovulatory and estrous animals, the apical cytoplasm contains secretory granules of at least three types. In addition, very irregularly shaped, dense, perinuclear granules occur, which may be another type of secretory granule or lysosomes. As compared to ciliated cells, the secretory cells have less prominent Golgi complexes, more abundant bundles of intermediate filaments, a more extensive glycocalyx on their apical surface, and more heterochromatic nuclei. In comparison to the cells of well-developed epithelia, the nonciliated cells of some other ovulatory and estrous rabbits are less well differentiated with fewer or no secretory granules and less well developed organelles. In the nonciliated cells of the long-term ovariectomized rabbits, there are no secretory or dense perinuclear granules. There is a decrease in the number of organelles that are involved in secretion, in the size of the cells, and in the amount of nuclear euchromatin.     

3α-Hydroxysteroid dehydrogenase and 3β-hydroxysteroid dehydrogenase in the ovary of young Mongolian gerbils

Summary The ovaries of sexually mature, pregnant mare serum gonadotropin (PMSG) stimulated, 12 week old Mongolian gerbils were investigated morphologically and enzyme histochemically for the appearance of the 3-hydroxysteroid and the 3-hydroxysteroid dehydrogenase during the estrous cycle. Up to ovulation, on day 3 of the estrous cycle, the number of vesicular follicles increases continuously. Primarily atretic follicles can be seen on day 4. On day 5 corpora lutea appear, but they degenerate already by day 6.During the entire estrous cycle, 3-hydroxysteroid dehydrogenase and 3-hydroxysteroid dehydrogenase activity can be found in the theca of tertiary follicles and in the interstitial cells, whereas the theca of secondary follicles and the granulosa of healthy follicles do not exhibit any enzyme activity. The activity decreases from day 1 till day 6. The granulosa of atretic follicles and the cells of corpora lutea show only weak activity. It may be significant that the intensity of enzyme activity in the ovary and the estrogen level in the plasma are differently correlated to the estrous cycle.This investigation was supported by the Deutsche Forschungsgemeinschaft     

Environmental factors influencing the seasonality of estrus in chimpanzees

Although the energetics of the estrous cycle in primates is not well understood, evidence suggests that energy and nutrient acquisition influence ovulation and the timing of conception. Energy for estrus has to compete with energy allocated for cellular maintenance, thermoregulation, movement for food, and predation avoidance. While some chimpanzee (Pan troglodytes) populations do not have a seasonal birth period, evidence suggests that there is seasonality in the number of estrous females. Similarly, the onset of postpartum cycles has been reported to be seasonal. We used 33 months of data from the Taï National Park, Côte dIvoire, to examine how the number of estrous females in a given month was influenced by the abundance and distribution of food, diet, rainfall and temperature. In a second analysis, we examined if there was a seasonal effect on first estrous swellings in adolescent females and postpartum adult females. Results demonstrated that the number of females in estrous in a given month was positively related to food abundance and percent foraging time spent eating insects, and negatively related to mean rainfall in the two preceding months and the mean high temperature. The timing of first estrous swellings of postpartum females and prepartum young females was positively related to the food abundance, and negatively related to mean high temperature. These results showed that environmental conditions can seasonally limit the energetically demanding estrus cycle. The presence of estrous females increases gregariousness in chimpanzee communities, and this study identified environmental factors that affect estrus directly and hence social grouping indirectly.     

Changes in expression of inhibin subunits in the cyclic golden hamster (Mesocricetus auratus) and the regulation of inhibin alpha subunit production by luteinizing hormone

In the present study, changes in localization of each inhibin subunit in the ovary were investigated during the estrous cycle of the golden hamster. The effect of LH surge on changes in localization in inhibin alpha subunit in the ovary was also investigated. Inhibin alpha subunit was localized in granulosa cells of various stages of follicles throughout the estrous cycle. Inhibin alpha subunit was also present in numerous interstitial cells on days 1 and 2 (day 1 = day of ovulation), but the number of positive interstitial cells was fewer on days 3 and almost disappeared on day 4 of the estrous cycle. Newly formed luteal cells were also positive for inhibin alpha subunit on days 1 and 2. On the other hand, positive reactions for inhibin beta A and beta B subunits were only present in the granulosa cells of healthy antral follicles. However, a positive reaction for inhibin beta B subunit in peripheral mural granulosa cells disappeared on days 3 and 4 of the estrous cycle. Treatment with LHRH-AS at 1100 h on day 4 completely blocked the luteinizing hormone (LH) surge and ovulation, although relatively high concentrations of plasma follicle-stimulating hormone (FSH) were maintained throughout the experiment. There were few positive reactions for inhibin alpha subunit in theca and interstitial cells 24 hr after LHRH-AS injection. The effect of LHRH-AS treatment was blocked by a single injection of 10 IU human chorionic gonadotropin. These results suggest that the major source of dimeric inhibin in the cyclic hamster was granulosa cells of healthy antral follicles. Different distribution pattern of inhibin beta A from beta B subunits in large antral follicles on days 3 and 4 of the estrous cycle suggests different secretion patterns of inhibin A from B on these days. Furthermore, the LH surge may be an important factor to induce production of inhibin alpha subunit in interstitial cells of the cyclic hamster.     

Effects of thymulin and GnRH on the release of gonadotropins by in vitro pituitary cells obtained from rats in each day of estrous cycle

The effects of thymulin and GnRH on FSH and LH release were studied in suspension cultures of anterior pituitary cells from female adult rats sacrificed on each day of the estrous cycle. The spontaneous release of gonadotropins by pituitaries, as well as their response to GnRH or thymulin addition, fluctuated during the estrous cycle. Adding thymulin to pituitary cells from rats in diestrus 1 increased the concentration of FSH; while in cells from rats in estrus, FSH level decreased. Thymulin had a stimulatory effect on the basal concentration of LH during most days of the estrous cycle. Adding GnRH increased FSH release in cells from rats in diestrus 1, diestrus 2, or proestrus, and resulted in higher LH levels in cells obtained from rats in all days of the estrous cycle. Compared to the GnRH treatment, the simultaneous addition of thymulin and GnRH to cells from rats in diestrus 1, diestrus 2, or proestrus resulted in lower FSH concentrations. Similar results were observed in the LH release by cells from rats in diestrus 1, while in cells from rats in proestrus or estrus, LH concentrations increased. A directly proportional relation between progesterone serum levels and the effects of thymulin on FSH release was observed. These data suggest that thymulin plays a dual role in the release of gonadotropins, and that its effects depend on the hormonal status of the donor's pituitary.     

The Expression Pattern of microRNAs in Granulosa Cells of Subordinate and Dominant Follicles during the Early Luteal Phase of the Bovine Estrous Cycle

This study aimed to investigate the miRNA expression patterns in granulosa cells of subordinate (SF) and dominant follicle (DF) during the early luteal phase of the bovine estrous cycle. For this, miRNA enriched total RNA isolated from granulosa cells of SF and DF obtained from heifers slaughtered at day 3 and day 7 of the estrous cycle was used for miRNAs deep sequencing. The results revealed that including 17 candidate novel miRNAs, several known miRNAs (n = 291–318) were detected in SF and DF at days 3 and 7 of the estrous cycle of which 244 miRNAs were common to all follicle groups. The let-7 families, bta-miR-10b, bta-miR-26a, bta-miR-99b and bta-miR-27b were among abundantly expressed miRNAs in both SF and DF at both days of the estrous cycle. Further analysis revealed that the expression patterns of 16 miRNAs including bta-miR-449a, bta-miR-449c and bta-miR-222 were differentially expressed between the granulosa cells of SF and DF at day 3 of the estrous cycle. However, at day 7 of the estrous cycle, 108 miRNAs including bta-miR-409a, bta-miR-383 and bta-miR-184 were differentially expressed between the two groups of granulosa cell revealing the presence of distinct miRNA expression profile changes between the two follicular stages at day 7 than day 3 of the estrous cycle. In addition, unlike the SF, marked temporal miRNA expression dynamics was observed in DF groups between day 3 and 7 of the estrous cycle. Target gene prediction and pathway analysis revealed that major signaling associated with follicular development including Wnt signaling, TGF-beta signaling, oocyte meiosis and GnRH signaling were affected by differentially expressed miRNAs. Thus, this study highlights the miRNA expression patterns of granulosa cells in subordinate and dominant follicles that could be associated with follicular recruitment, selection and dominance during the early luteal phase of the bovine estrous cycle.     

Papanicolaou staining of exfoliated vaginal epithelial cells facilitates the prediction of ovulation in the giant panda

The giant panda is seasonally monoestrus, experiencing a single estrous with spontaneous ovulation in the spring. Therefore, accurate monitoring of the estrous cycle to pinpoint the time of ovulation is critical for the success of timed mating or artificial insemination. Analysis of exfoliated vaginal epithelial cells is a simple technique that rapidly yields information about the estrous status of a panda. Vaginal swabs were obtained during five estrous cycles of two nulliparous females. Cells were stained with the trichrome Papanicolaou and classified as basophils, intermediates or superficials. The color of stained cells, basophilic, acidophilic or keratinized, was recorded as a characteristic independent of the three standard cell types. The day urinary conjugates of estrogen fell from peak levels was considered the day of ovulation. A chromic shift occurred 8-9 days before ovulation when the majority of exfoliated vaginal cells changed from basophilic (blue) to acidophilic (pink) without accompanying nuclear or cytoplasmic changes. A second chromic shift was consistently observed 2 days prior to ovulation when keratinized (orange) cells replaced acidophils as the majority of vaginal cells. Monochrome staining of vaginal cells is sufficient to quantify superficial cells, which is a useful adjunct to behavioral and endocrinological data in determining estrous in the giant panda. However, the timing and duration of superficial cell elevations are substantially different between and within individual females, which limits the accuracy of timing ovulation for artificial insemination. The predictive value of vaginal cytology was greatly enhanced with the trichrome stain and evaluation of cell color.     

Evidence for "memory" of cervical stimulation for the promotion of pregnancy in rats

Successful pregnancy in the female rat depends upon two sets of physiological events: (1) transport of gametes (sperm and egg) through the reproductive tract so that fertilization can be effected and (2) establishment of an appropriate hormonal environment (progestational state) so that the fertilized egg can implant in the uterus and be maintained during subsequent gestation. This study highlights the independence of the mechanisms controlling the gametic and hormonal aspects of pregnancy by temporally separating the introduction of sperm into the female from the stimulation that triggers the progestational hormonal response. The progestational state was initiated by electrical stimulation of the cervix, and sperm was introduced directly into the uterus by artificial insemination. Although these two events were separated by up to 3 days, pregnancy could ensue. Cervical stimulation, normally a consequence of male intromission behavior, establishes a condition in the central nervous system, a "memory" that signals the probable induction of pregnancy. Without this "memory," animals with a short estrous cycle would continue to cycle after mating, thereby producing a hormonal environment incompatible with implantation. The "memory" is manifested by daily surges of prolactin irrespective of fertilization. This is the first physiological demonstration that a "memory" of cervical stimulation can be called upon to support a viable pregnancy.     

Estrous cycle regulation in the whitefooted mouse (Peromyscus leucopus) with special reference to vaginal cast formation

The estrous cycle of the whitefooted mouse (Peromyscus leucopus) was consistent with those described for most small rodents and closely resembled that of the house mouse and the rat in timing. The presence of the male did not accelerate puberty nor induce cycle synchrony, but was critical in establishing cycle regularity. Females isolated from influences of males never attained a pattern of concistent cycle length. Development of the vaginal epithelium during estrus was excessive in both isolated and non-isolated females, resulting in a "cast" of cornified epithelium which could be removed intact during metestrus. Casts removed during the making of vaginal smears measured up to 20 mm in length and still left a sufficient residue of cells which could be detected in all stages of the estrous cycle.     

Olfactory preferences, scent marking, and "proceptivity" in female hamsters

Two appetitive sexual behaviors of female hamsters, attraction to odors of males and vaginal scent marking, were investigated and the correlations of each to reproductive states were determined. Both estrous and diestrous females were more attracted to the odors of intact males than to the odors of females or castrated males; this differential attraction was more pronounced for estrous females. Lactating females, but not pregnant females, were also preferentially attracted to odors of intact males. Thus, odors alone provided sufficient information to allow sexual discrimination by females, and estrous, diestrous, and lactating females were preferentially attracted to odors of intact males whereas pregnant females were not. Reproductive condition had no influence on responses to odors of castrated males or diestrous females. Vaginal scent marking was strongly influenced by reproductive state: Marking frequency was low in estrous, pregnant, and early lactation females, but high in late lactation and diestrous females, suggesting a function of advertisement of approaching receptivity. Thus sexual preferences based on olfactory cues and vaginal scent-marking frequency differed in their relationships to reproductive conditions. It is suggested that in other species such relationships between appetitive sexual behaviors and reproductive condition will depend on the species social organization, ecological niche, and taxonomic group, and consequently that the concept of preceptive behaviors should be broadened to include all appetitive sexual behaviors of females, not just those that are correlated with the hormonal state characterizing estrus.     

Expression of aquaporin 1 in the pig peri-ovarian vascular complex during the estrous cycle and early pregnancy

Aquaporin 1 (AQP1) is a water channel protein expressed in endothelial and epithelial cells of many tissues, including the vasculature, where it serves to increase water permeability of the cell membrane. The aim of this study was to investigate the expression and distribution of AQP1 in porcine peri-ovarian vascular complex (PVC) during the estrous cycle and early pregnancy. Immunohistochemistry and semi-quantitative immunoblotting techniques were used. We have demonstrated the presence of AQP1 protein in the endothelial cells of the lymphatic and vascular endothelium of the PVC during the pig estrous cycle and early pregnancy. The expression of AQP1 protein in the PVC did not change significantly between Days 10-12 and 14-16, but increased on Days 2-4 and 18-20 when compared with Days 10-12 and 14-16 of the estrous cycle. In pregnant gilts, the expression of AQP1 did not differ significantly during the onset and the end of the implantation process and also when compared to the mid- and late-luteal phases of the estrous cycle. In conclusion, AQP1 is expressed in the endothelial cells of PVC and may modulate hormonal regulation of reproductive organs.     

PGE receptor characteristics on porcine luteal cells during the estrous cycle and early pregnancy.

This study examined the affinities and concentrations of prostaglandin E (PGE) receptors on porcine luteal cells during the estrous cycle and early pregnancy. Corpora lutea (CL) were obtained from nonpregnant gilts at days 9 (n = 4), 12 (n = 3), and 14 (n = 6); three gilts possessed red, vascular CL and three gilts had white nonvascular CL) of the estrous cycle, and days 9 (n = 4), 12 (n = 3), 14 (n = 5), and 30 (n = 5) of pregnancy. The CL were dissociated enzymatically to disperse single cells and the red blood cells were removed by elutriation. The luteal cells were assayed for specific PGE binding by displacement analysis with use of [3H] PGE2 and varying concentrations of unlabeled PGE2. The specific binding of [3H] PGE2 to luteal cells decreased (p < 0.05) from days 9 to 14 of the estrous cycle, but only decreased (p < 0.05) from days 9 to 12 of pregnancy. Specific binding was higher (p < 0.05) on day 14 of pregnancy than the comparable stage of the estrous cycle. The affinities of PGE receptors decreased (p < 0.05) only on the luteal cells dissociated from red, vascular CL of day 14 nonpregnant gilts compared with those of other days of the estrous cycle and pregnancy. The number of PGE receptors on porcine luteal cells was similar (p > 0.05) in pregnant and nonpregnant gilts, but decreased (p < 0.05) on days 12-14 postestrus. During early pregnancy, it was evident that high affinity PGE receptors are sustained on porcine luteal cells; however, the role of the PGE receptors in maternal recognition of pregnancy remains speculative.     

Morphometric analysis of cell types in the ovine corpus luteum throughout the estrous cycle

The cellular composition of ovine corpora lutea obtained during the early (Day 4), mid (Days 8 and 12), and late (Day 16) stages of the estrous cycle was determined by morphometric analysis. Individual corpora lutea were collected via midventral laparotomy from a total of 19 ewes. A center slice from each corpus luteum was processed for electron microscopy and subsequent morphometric analysis of the numbers and sizes of steroidogenic and nonsteroidogenic cells. Luteal weight progressively increased throughout the estrous cycle (p less than 0.05). Corpora lutea collected on Day 16 were assigned to one of two subgroups on the basis of gross appearance and weight: nonregressed (NR, 542 +/- 25 mg) or regressed (R, 260 +/- 2 mg). There were no significant changes in the proportion of the corpus luteum occupied by small luteal cells (19 +/- 2%) or large luteal cells (36 +/- 1%) throughout the estrous cycle. The total number of steroidogenic cells per corpus luteum increased from 21.8 +/- 3.7 (X 10(6)) on Day 4 to 61.7 +/- 5.4 (X 10(6)) on Day 8 (p less than 0.05) and remained elevated thereafter. The number of small luteal cells was 10.0 +/- 2.7 (X 10(6)), 39.7 +/- 1.4 (X 10(6)), 46.1 +/- 5.8 (X 10(6)), 49.0 +/- 13.7 (X 10(6)), and 29.9 +/- 8.6 (X 10(6)) on Days 4, 8, 12, 16 (NR), and 16 (R), respectively (p less than 0.05, Day 4 vs. Days 8, 12, 16 NR). In contrast, the number of large luteal cells was 11.8 +/- 1.5 (X 10(6)) on Day 4 and did not vary significantly during the remainder of the estrous cycle. The numbers of nonsteroidogenic cell types increased (p less than 0.05) from Day 4 to Day 16 (NR) but were decreased in regressed corpora lutea (Day 16 R). Regression was characterized by a 50% decrease (p less than 0.05) in the total number of cells per corpus luteum from 243 +/- 57 ( X 10(6)) on Day 16 (NR) to 125 +/- 14 ( X 10(6)) on Day 16 (R) (p less than 0.05). Small luteal cells remained constant in volume throughout the entire estrous cycle (2520 +/- 270 microns 3), whereas large luteal cells increased in size from 5300 +/- 800 microns 3 on Day 4 to 16,900 +/- 3300 microns 3 on Day 16 (NR) (p less than 0.05). In summary, small luteal cells increased in number but not size throughout the estrous cycle, whereas large luteal cells increased in size but not number.     

Fluctuation of Aquaporin 1, 5, and 9 Expression in the Pig Oviduct during the Estrous Cycle and Early Pregnancy

Thirteen mammalian aquaporin (AQPs) isoforms with a unique tissue-specific pattern of expression have been identified. To date, 11 isoforms of AQP have been reported to be expressed in female and male reproductive systems. The purpose of our study was to determine the localization and quantitative changes in the expression of AQP1, 5 and 9 within the pig oviduct during different stages of the estrous cycle and early pregnancy. The results demonstrated that AQP1, 5, and 9 were clearly detected in all studied stages of the estrous cycle and pregnancy. AQP1 was localized within oviductal blood vessels. In cyclic gilts, the expression of AQP1 protein did not change significantly between days 10–12 and 14–16 but increased on days 2–4 and 18–20. AQP5 was localized in smooth muscle cells and oviductal epithelial cells. The expression of AQP5 protein did not change significantly between days 10–12 and 14–16 of the estrous cycle but increased on days 2–4 and 18–20. The anti-AQP9 antibody labeled epithelial cells of the oviduct. The expression of AQP9 did not change significantly between days 10–12 and 14–16 of the estrous cycle but increased on days 2–4 and 18–20. In pregnant gilts, expression of AQP1, 5, and 9 did not change significantly in comparison with the estrous cycle. Therefore, a functional and distinctive collaboration seems to exist among diverse AQPs in water handling during the different oviductal phases in the estrous cycle and early pregnancy.     

3 Alpha-hydroxysteroid dehydrogenase and 3 beta-hydroxysteroid dehydrogenase in the ovary of young Mongolian gerbils

The ovaries of sexually mature, pregnant mare serum gonadotropin (PMSG) stimulated, 12 week old Mongolian gerbils were investigated morphologically and enzyme histochemically for the appearance of the 3 alpha-hydroxy-steroid and the 3 beta-hydroxysteroid dehydrogenase during the estrous cycle. Up to ovulation, on day 3 of the estrous cycle, the number of vesicular follicles increases continuously. Primarily atretic follicles can be seen on day 4. On day 5 corpora lutea appear, but they degenerate already by day 6. During the entire estrous cycle, 3 alpha-hydroxysteroid dehydrogenase and 3 beta-hydroxysteroid dehydrogenase activity can be found in the theca of tertiary follicles and in the interstitial cells, whereas the theca of secondary follicles and the granulosa of healthy follicles do not exhibit any enzyme activity. The activity decreases from day 1 till day 6. The granulosa of atretic follicles and the cells of corpora lutea show only weak activity. It may be significant that the intensity of enzyme activity in the ovary and the estrogen level in the plasma are differently correlated to the estrous cycle.     

Regulation of the estrous cycle by neutrophils via opioid peptides

We found previously that neutrophil-depleted mice exhibited significant blockading of both the regular estrous cycle and cyclic changes of steroid hormone levels. In this study, we aimed at elucidation of the underlying mechanism. To examine the possibility that an increase in bacteria in the vaginal vault of neutrophil-depleted mice causes blockading of the estrous cycle, we treated neutrophil-depleted mice with antibiotics but failed to restore the estrous cycle. We then examined another possibility that neutrophils regulate the estrous cycle via opioid peptides, because opioid peptides regulate steroidogenesis in theca and granulosa cells in the ovaries, and because neutrophils contain opioid peptides. In support of this possibility, naloxone, an opioid antagonist, blocked the estrous cycle and a μ opioid receptor agonist restored the estrous cycle in neutrophil-depleted mice. Pro-opiomelanocortin was immunohistochemically detected in peripheral blood neutrophils but not in ones that had infiltrated into the ovaries. i.v. injection of anti-MIP-2 polyclonal Ab caused blockading of the estrous cycle, whereas MIP-2 was detected in the ovaries, suggesting a role of MIP-2 in the regulation of the estrous cycle. Moreover, i.v. injection of MIP-2 decreased the pro-opiomelanocortin signal in peripheral blood neutrophils and caused blockading of the estrous cycle. Together, these results suggest that neutrophils maintain the estrous cycle via opioid peptides.