Immunocytochemical localization of neurophysin and oxytocin in ovine corpora lutea

The cellular distribution of neurophysin and oxytocin within ovine corpora lutea obtained on Days 4, 10 and 16 of the estrous cycle was examined immunocytochemically. Serial sections (8-10 micron-thick) prepared from corpora lutea that had been fixed in Bouin's solution and embedded in paraffin were immunostained for neurophysin or oxytocin using the peroxidase-antiperoxidase (PAP) procedure. Irrespective of the day of the cycle examined, immunoreactivity was restricted to large luteal cells. However, on Days 4 and 10 of the cycle, the intensity of staining in large luteal cells was highly variable; and, within the same section some cells were heavily stained, others were only lightly stained, and still others were not stained at all. In contrast, on Day 16 of the cycle, the intensity of staining was uniform and essentially all of the large luteal cells were immunoreactive. Based on the results obtained, it is evident that immunoreactive neurophysin and oxytocin can be detected as early as Day 4 of the cycle, persists through Day 15, and is restricted to large luteal cells.     

Variations in oxytocin, vasopressin and neurophysin concentrations in the bovine ovary during the oestrous cycle and pregnancy

Bovine ovaries were obtained from the abattoir and corpora lutea were classified as: (1) early luteal phase (approximately Days 1-4); (2) mid-luteal phase (Days 5-10); (3) late luteal phase (Days 11-17); (4) regressing (Days 18-20) and (5) pregnant (Days 90-230). In addition, preovulatory follicles and whole ovaries without luteal tissue were collected. Concentrations of oxytocin, vasopressin, bovine neurophysin I and progesterone were measured in each corpus luteum by radioimmunoassay. Progesterone and neurophysin I levels increased from Stage 1 to Stage 2, plateaued during Stage 3 and declined by Stage 4. Oxytocin and vasopressin concentrations increased from Stage 1 to Stage 2 but declined during Stage 3 and were low (oxytocin) or undetectable (vasopressin) in follicles, whole ovaries and pregnancy corpora lutea. Therefore the concentrations of both peptide hormones were maximal during the first half of the cycle and declined before those of progesterone. The high concentration of oxytocin within the corpus luteum coupled with the presence of bovine neurophysin I suggests that oxytocin is synthesized locally.     

Immunocytochemical evidence for the presence of oxytocin and neurophysin in the large cells of the bovine corpus luteum

Summary The presence of neurophysin, oxytocin and vasopressin in the bovine corpus luteum was examined immunocytochemically. Tissue blocks of corpora lutea from pregnant and non-pregnant animals were fixed with glutaraldehyde/paraformaldehyde fixative and immunostained by the peroxidase-antiperoxidase (PAP) method. The simultaneous presence of immunoreactive oxytocin and immunoreactive oxytocin-neurophysin was demonstrated in large luteal cells of non-pregnant animals, while no staining for vasopressin or vasopressin-neurophysin was observed. None of the peptides were detected in the corpus luteum of pregnant animals. The small luteal cells were not found to be stainable at any time.     

Oxytocin in baboon (Papio anubis) corpus luteum

Recently, using a highly specific radioimmunoassay, we have demonstrated that the concentration of oxytocin in the corpus luteum of the human and cynomolgus monkey are several fold higher than in the peripheral circulation. In this study, we have examined the corpora lutea and ovarian stroma from the ovaries of normal adult cycling baboons (Papio anubis) for the presence of oxytocin through the use of immunocytochemical procedures. Tissues obtained at laparotomy were fixed in Bouin's solution and embedded in paraffin; immunoreactive oxytocin was localized with peroxidase-antiperoxidase and 3.3' diaminobenzidine. Six corpora lutea with stroma were obtained--two each from the early (Day 14-20), mid-(Day 21-24), and late (Day 25-30) stages of the luteal phase. Immunoreactive oxytocin was localized in all corpora lutea examined but was absent from all stroma samples. Larger areas of the corpus luteum from the mid-luteal phase showed staining for oxytocin, and the intensity of staining for this peptide was maximal in this phase of the cycle.     

Characterization of large luteal cells and their secretory granules during the estrous cycle of the cow.

Large steroidogenic cells of the bovine corpora lutea were evaluated for morphological changes on Days 3, 7, 11, 14, 17, and 19 of the estrous cycle. Large cells were readily identified by size (25-50 microns diameter), numerous mitochondria, and the presence of dense secretory granules (150-300 nm in diameter). These granules were found in a discrete cluster and were not dispersed throughout the cytoplasm. Only 3% of the large cells contained a cluster of granules on Day 3. The percentage was highest during midcycle (Day 7, 84%; Day 11, 64%), dropped on Day 14 (26%), and was lowest on Days 17 (16%) and 19 (8%). Electron microscopic immunocytochemistry showed that oxytocin and neurophysin were co-localized in these granules on all days evaluated. As early as Day 14, large cells were observed with characteristics typical of regressing corpora lutea, i.e., a reduction in cells with secretory granules, large cytoplasmic lipid droplets, and swollen mitochondria with dense inclusions. However, since this was a time of the cycle when plasma concentrations of progesterone were very high, this corpus luteum is referred to as involutive rather than regressive. Our results may be summarized as follows: 1) from Day 7 to Day 14 there was a 69% decline in the number of large cells containing oxytocin-laden secretory granules. This occurred prior to the rise in uterine oxytocin receptors and the large luteolytic pulses of prostaglandin that reportedly occur after Day 14. The role of this apparent early release of oxytocin is not known. 2) Large steroidogenic luteal cells of the estrous cycle have morphological characteristics similar to those of large luteal cells during pregnancy. However, large luteal cells of the estrous cycle contain oxytocin whereas those of pregnancy are devoid of oxytocin.     

Evidence for the secretion of immunoreactive neurophysin I in addition to oxytocin from the ovary in cattle

In Exp. I, blood samples were collected simultaneously from the posterior vena cava and jugular vein or aorta from 7 heifers every 5-20 min for 2-5 h. Concomitant pulsatile secretion of oxytocin and immunoreactive neurophysin I was detected in the vena cava, but not in the jugular vein or aorta. Concentrations of oxytocin and immunoreactive neurophysin increased earlier and were higher in the vena cava than in the jugular vein or aorta after the injection of a luteolytic dose of prostaglandin F-2 alpha analogue during the mid-luteal phase of the oestrous cycle, demonstrating its ovarian but not pituitary origin. In Exp. II, blood samples were collected from the jugular vein every 12 h during 1 week after oestrus. Follicular growth had been stimulated during the preceding oestrous cycle with PMSG (10 heifers and cows) or with FSH (5 animals); 6 heifers served as controls. There was a high correlation between the number of follicles or CL and the increase in oxytocin and immunoreactive neurophysin I. Although PMSG had a greater luteotrophic effect than did FSH on progesterone secretion, a similar stimulation of oxytocin and immunoreactive neurophysin I was not observed. It is concluded that immunoreactive neurophysin I and oxytocin are secreted from the ovary in concentrations dependent upon the number of corpora lutea (and of follicles) present. During the mid-luteal period the secretion occurs in a concomitant pulsatile fashion.     

Demonstration of luteinizing hormone/human chorionic gonadotrophin receptor-binding inhibitor in aqueous extracts of frozen human corpus luteum

Aqueous extracts of frozen human corpora lutea were tested for the presence of an inhibitor of luteinizing hormone-receptor site binding (LHRBI) and for the subsequent effect on the stimulatory response of luteinizing hormone (LH) on progesterone synthesis by sheep ovarian cells. In the presence of human corpus luteum extract of normal menstrual cycle (30,000-g supernatant), the binding of 125I human chorionic gonadotrophin (hCG) to granulosa and luteal cells of sheep ovaries was markedly reduced, but the ability of rat testicular LH receptors to bind labelled hCG was less affected. However, extracts of corpora lutea of the first trimester of pregnancy appeared to be less inhibitory on the binding of LH/hCG to ovarian cells and had no effect on the binding of rat testicular cells compared to those of normal menstrual cycle. Addition of both extracts separately inhibited the LH-stimulated in vitro progesterone synthesis by granulosa cell cultures and by incubated sheep corpus luteum slices. These findings provide evidence for the presence of LHRBI in human corpus luteum.     

Ovulation in the merino ewe in the breeding and anoestrous seasons

The pattern of ovulation of Merino ewes was studied by repeated laparoscopy each 14 days in the anoestrous (n = 97) and breeding (n = 87) seasons. In the anoestrous season the proportion of ewes ovulating did not decrease below 11%, 42% of ewes never ovulated and the remainder fluctuated between the two states. On 20 occasions a clear anovulatory period was interrupted by an isolated spontaneous ovulation. In the breeding season the overall mean proportion of ewes with corpora lutea or albicantia at laparoscopy was 87%, 54% of ewes ovulated regularly throughout while in another 31% absence of corpora lutea or albicantia coincided with the follicular phase of an oestrous cycle as evidenced by an appropriately aged corpora lutea at the next laparoscopy. Of the remaining 15% of the flock 3% had anovulatory periods greater than 14 days while the remainder experienced irregular ovulatory cycles--the majority due to short periods of anovulation but some ewes retained corpora lutea for longer than 14 days while others ovulated twice between successive laparoscopies.     

Reproduction in female Hartmann's zebra, Equus zebra hartmannae

Ovaries, fetuses and plasma were collected from zebra mares shot in the Etosha National Park in Namibia between 15 and 25 August 1983. Ovarian weight was affected by reproductive status and most of the non-pregnant mares were anoestrous. The number of follicles varied between individuals and only pro-oestrous/oestrous mares had follicles larger than 20 mm in diameter. The largest follicle in pregnant mares was only 9 mm in diameter. Corpora lutea and corpora albicantia were found in non-pregnant as well as pregnant mares: 4 pregnant mares had only corpora albicantia. The presence of secondary corpora lutea could not be confirmed in any of the pregnant mares. Implantation was estimated to occur at around 73 days of gestation, and most mares (84%) had conceived between November and April. Peripheral concentrations of plasma progesterone during pregnancy varied from 0.5 to 2.4 ng/ml.     

Human corpus luteum: Immunocytochemical evidence for presence of prolactin

Summary Six human corpora lutea (day 17–25) of the menstrual cycle and 4 ovarian stromal tissues from 7 cycling women were examined for the presence of the hormone, prolactin, by immunohistochemistry using the indirect peroxidase-antiperoxidase method. After mounting tissue sections of 4 m, endogenous peroxidases were removed with hydrogen peroxide and the sections were incubated for l h at room temperature followed by 16 h at 4° C with a highly specific antisera for human prolactin, nonimmunized normal rabbit serum for a control reaction, or antiserum preadsorbed with excess human prolactin for specificity determination. Following the reaction with the second antibody (goat antirabbit IgG) for l h at room temperature, prolactin was localized using peroxidase anti-peroxidase and 3.3-diaminobenzidine as the chromogen. Prolactin was present and could be localized in the luteal cells of all 6 corpora lutea, but not in any of the ovarian stroma studied. Human adenohypophysis served as a positive tissue control for prolactin immunopositive staining. The localization of immunoreactive prolactin in the corpus luteum demonstrates directly the presence of this hormone in the human ovary, adding further evidence for its role in luteal function.     

Absence of specific follicle stimulating hormone receptors in porcine corpora lutea

Crude cell membrane preparations of corpora lutea from 13 pigs in different phases of the estrous cycle or pregnancy were assayed for the presence of specific follicle stimulating hormone (FSH) receptors using highly purified ovine FSH. Testicular tissue from boars and granulosa cells from porcine follicles served as positive controls. Scatchard analysis was used to determine binding affinity of FSH to target tissues as well to study human chorionic gonadotropin (hCG) bindability to corpora lutea membranes. In contrast to testicular tissue and granulosa cells, no specific FSH binding was detected in luteal tissue during the estrous cycle or pregnancy in pigs.     

Localization of androgen receptor in the follicle and corpus luteum of the primate ovary during the menstrual cycle

Ovarian androgens may act locally to modulate follicular and luteal function in various species. This study examined the distribution of androgen receptors within the primate ovary throughout the menstrual cycle. Ovaries were collected from rhesus and cynomolgus monkeys during the early, mid-, and late (n = 3-5 per stage) follicular and luteal phases of the cycle. The tissues were processed for indirect immunocytochemical localization of androgen receptors with a specific monoclonal antibody against human androgen receptor (AN1-15). In addition, ovaries (n = 3) were collected from rhesus monkeys for biochemical detection of androgen receptor using 3H-androgen and AN1-15. Specific immunocytochemical staining, as determined by comparing adjacent tissue sections incubated with either AN1-15 or a nonspecific control antibody, was exclusively nuclear. Androgen receptor was detected in the germinal epithelium and ovarian stroma at all stages of the cycle. The thecal and granulosa cells of growing follicles, and of many but not all atretic follicles, contained androgen receptors. Luteinizing granulosa cells of the periovulatory follicle and luteal cells from the early and midluteal phase stained intensely for androgen receptor. Regressing corpora lutea of the late luteal phase also stained for androgen receptor; however, fully regressed corpora lutea in the early follicular phase of the next cycle did not exhibit receptor staining. Luteal cells that were androgen receptor-positive also stained histochemically for the presence of 3 beta-hydroxysteroid dehydrogenase. Sucrose gradient analysis with radiolabeled androgen demonstrated a shift in the androgen receptor peak in monkey ovarian tissue upon addition of AN1-15, confirming the presence of androgen receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical localization of relaxin in human corpora lutea: cellular and subcellular distribution and dependence on reproductive state

Relaxin is one of the hormones present during pregnancy and it is synthesized primarily by corpora lutea (CL). Other reproductive tissues including CL of the menstrual cycle may also synthesize this hormone. Very little is known, however, about the cellular and subcellular distribution of relaxin in human CL and dependence of luteal relaxin on the reproductive state. The light and electron microscope immunocytochemical studies described here were undertaken to obtain this information using antisera to porcine and human relaxin. Immunostaining was found in large luteal cells (17-30 microns) but not in small luteal cells (7-16 microns) or in nonluteal cells in any of the reproductive states or in human hepatocytes. Luteal immunostaining was low in early luteal phase; it increased progressively, reaching the highest level in late luteal phase, and then decreased greatly in corpora albicantia. Term pregnancy CL contained similar immunostaining as early luteal phase CL. Mid luteal phase CL contained more immunostained cells than late luteal phase CL, but the late luteal phase CL contained a greater amount of immunostaining per cell than mid luteal phase CL. The immunogold particles due to relaxin were primarily present in secretory granules and to a small extent in rough endoplasmic reticulum. Quantitation revealed that secretory granules contained a much higher number of gold particles than did rough endoplasmic reticulum. These two organelles from late luteal phase CL contained greater numbers of gold particles than those from mid luteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell-specific localization of progesterone receptors in the bovine ovary at different stages of the oestrous cycle

This immunohistochemical study describes the localization of progesterone receptors (PR) in the bovine ovary of 23 cows at different stages of the oestrous cycle. In primordial, primary and secondary follicles the score for PR in the follicle cells increased progressively with the maturation of the follicle. In vital tertiary follicles and cystic atretic follicles a moderate score for PR was found, while in obliterative atretic follicles the score was much lower. Scores were high in corpora hemorrhagica, low in corpora lutea and still lower in corpora albicantia. Low PR scores were also found in the tunica albuginea and surface epithelium. Cyclic variations of PR immunoreactivity were manifest in most ovarian tissues. Follicular scores for PR were high in oestrus and decreased during the following stages, whereas scores in corpora lutea cells varied according to a characteristic pattern with high levels during oestrus and metoestrus. The variations in the scores for PR in the different ovarian cell types suggest a cell-specific and cycle-dependent influence of progesterone. A negative correlation was found between the PR scores and the plasma progesterone concentration.     

Neurophysin in the large luteal cell of the nonpregnant water buffalo (Bubalus bubalis): immunohistochemical localization

Light microscopy immunohistochemistry was used to localize neurophysin in the corpus luteum of the mid-luteal phase of the estrous cycle of the water buffalo (Bubalus bubalis). Corpora lutea weighing 0.39-0.65 g from a recent ovulation showed no staining. Corpora lutea identified with the late luteal phase showed only weak evidence of staining. The neurophysin staining was confined to a specific region of large oval-shaped cells (20-30 microns diameter), which had a very eosinophilic cytoplasm. The intense localization of staining to a distinct area of the cytoplasm was previously only observed in the corpus luteum of the cow. Corpora lutea obtained from all quadrants of pregnancy did not stain. Controls in which the neurophysin antiserum was substituted with serum from an unimmunized rabbit (normal rabbit serum) or neurophysin antiserum preabsorbed with bovine oxytocin-associated neurophysin I also did not stain. These data indicate the neurophysin is present in the mature corpus luteum of the nonpregnant water buffalo as it is in other nonpregnant ruminants, the ewe and cow.     

Radioimmunoassay of progesterone, 17-hydroxyprogesterone, estradiol-17beta and prostaglandin F in human corpus luteum.

Radioimmunoassay procedures have been adapted for the assay of progesterone, 17-hydroxyprogesterone, estradiol-17beta, and prostaglandin F in human corpus luteum. The method utilises a single homogenisation and extraction of the tissue followed by fractionation of the steroids on alumina, and separation of the prostaglandins of the F series from the E and A series on silica gel, prior to radioimmunoassay. An attempt has been made to validate the method for the progestins by comparison with results after fractionation of the progestins on Sephadex LH-20, for estradiol-17beta by comparison with values obtained with competitive protein-binding, and for prostaglandin F by comparison with values after additional purification. The results showed that peak concentrations of the three steroids in corpora lutea from women during the luteal phase of the menstrual cycle were comparable to those found in corpora lutea from women in early pregnancy. However, in six out of fourteen corpora lutea from non-pregnant women, prostaglandin F levels were higher than those found in corpora lutea from seven women in early pregnancy, i.e. 13-46 ng/g compared with 1-7 ng/g. Of the above six corpora lutea, four were on days 23-25 of the cycle, at a time when luteolysis would be commencing. The results in this paper support the conclusion that the corpus luteum is a major site of synthesis of the three steroids examined, although the site of synthesis of prostaglandin F is still equivocal.     

The conversion of pregnenolone-7alpha-3H and progesterone-4-14C to estrogens by corpora lutea of menstrual cycles and pregnancy.

The metabolism of pregnenolone-7alpha-3H and progesterone-4-14C by human corpora lutea tissue of menstrual cycles and pregnancy was studied. In the incubations, equimolar mixtures of pregnenolone-7alpha-3H and progesterone-4-14C were used as substrates. Three corpora lutea of cycles were used as minced tissue. From those corpora lutea progesterone, 17-hydroxyprogesterone and androstenedione were formed, although no estrogens were formed. One corpus luteum of cycle and one corpus luteum of pregnancy were used as homogenated tissue, and those formed estrone and estradiol as well as the same three delta4-metabolites. The corpus luteum of cycle also formed testosterone. All metabolites including estrogens showed the lower 3H to 14C ratio than the starting ratio. 17-hydroxypregnenolone in only one corpus luteum, and no delta5-metabolites in the other four corpus luteum were identified. It is therefore proposed that the major pathway for estrogen formation in human corpus luteum is pregnenolone yields progesterone yields 17-hydroxyprogesterone yields androstenedione (or testosterone) yields estrone and estradiol.     

Carbon-13 nuclear magnetic resonance studies of the binding of selectively 13C-enriched oxytocins to the neurophypophyseal protein, bovine neurophysin II

Complex formation between bovine neurophysin II and oxytocin molecules containing 85% 13C enrichment in specific amino acid residues was studied using 13C nuclear magnetic resonance spectroscopy. Chemical shift and relaxation time values of the analogue [13C-Leu3]oxytocin, [13C-Gly9]oxytocin, and the doubly labeled [13C-Ile3 Gly9]oxytocin were obtained for the hormones in the absence and presence of neurophysin. The results showed that certain 13C nuclear magnetic resonance parameters of residue 3 but not of residue 9 of oxytocin are altered upon binding to neurophysin. These observations suggest that residue 3 but not residue 9 is involved in the protein-hormone interaction and they demonstrate the general applicability of selective 13C enrichment for the study of peptide-protein interactions.     

Age-related changes in ovarian morphology from birth to menopause in the Japanese monkey,Macaca fuscata fuscata

Age-related changes in ovarian morphology were studied in female Japanese monkeys,Macaca fuscata fuscata. A total of 47 nonlactating females of various ages ranging from new-born to >28 yr old were used. Ovarian size increased during the first decade of life, reached a plateau at around 10 yr. This was followed by a gradual decline throughout the remaining life span. The ovarian cortex of new-born animals consisted of numerous clusters of mitotic primordial germ cells. Such mitotic germ cells were observed even in the ovary of 28-day-old animal, but were not found in any animal after 1.5 yr of age. Numbers of primordial follicles decreased exponentially with the advance of age, and only a few primordial follicles were observed after about 16 yr of age. The numbers of primary and tertiary follicles increased from ages 4 to 16 yr, with a peak at 8 to 10 yr, and then decreased gradually. Developing tertiary follicles were observed as early as 1.5 yr of age. About 40% of tertiary follicles were atretic follicles throughout life, and their size was similar to that of developing tertiary follicles. Corpora lutea or corpora albicantia were found in ovaries more than 4 yr old. Remnants of corpora lutea and corpora albicantia, together with thick-walled blood vessels and fibrosis, became apparent in ovaries after 16 yr, and were observed characteristically in ovaries over 26 yr of age. There was no significant difference in the number or in the size of tertiary follicles between the breeding and nonbreeding seasons.     

Concentration of prostaglandins PGE and PGF, estrone, estradiol, and progesterone in human corpora lutea

The concentrations of prostaglandins PGE and PGF, estrone, estradiol and progesterone in human corpora lutea were measured by radioimmunoassay at various stages of the luteal phase of the menstrual cycle. The concentrations of PGF were found to be significantly higher in both the mid and late luteal phases than in the early luteal phase. In the mid luteal phase there was a concomittant increase in PGE levels, but these levels had declined in the late luteal phase. Steroid concentrations were generally lower in the late luteal phase.

It has been postulated that in the human corpus luteum locally produced prostaglandins may be responsible for luteolysis. Our data on the concentrations of PGF and PGE in corpora lutea at various stages of the luteal phase support such a possibility.