Hormonal behavior correlates with follicular recruitment at mid-gestation in the South American plains vizcacha, Lagostomus maximus (Rodentia, Caviomorpha).

In mammals, hormonal regulation during gestation is crucial for embryo implantation and pregnancy success. This regulation is controlled through the level of progesterone (P4) that blocks the activity of the hypothalamic-hypophyseal-gonadal (HHG) axis. Previous studies in the pregnant South American plains vizcacha, Lagostomus maximus, have shown that the HHG axis activates around mid-gestation, promoting pre-ovulatory follicle formation. However, the characterization of the hormonal dynamics throughout gestation and its ovarian correlation has not been studied in depth. We studied the ovarian dynamics of L. maximus and its correlation with the hormonal profile during gestation, analyzing serum levels of P4, 17ß-estradiol (E2), 4Δ-androstenedione (A4), luteinizing hormone (LH) and follicle stimulating hormone (FSH) as well as the ovarian distribution and expression of their receptors. Additionally, we have analyzed the folliculogenesis and accessory corpora lutea (ACL) formation. P4 showed two concentration peaks reaching its highest level at mid-gestation decreasing at 91-100days post-coitum. P4 decrease is followed by an increase of circulating levels of A4, E2, FSH and LH and with an elevated number of antral/pre-ovulatory follicles which express PGR, ESR1, ESR2, AR, LHR and FSHR. In addition, ACL with oocyte retention and cytoplasmic lipid droplets in luteal cells were detected at this time point. These results show that in L. maximus the decrease of P4 level from mid-gestation enables follicular recruitment until pre-ovulatory stage and the development of functional ACL.

A Novel Intravital Imaging Window for Longitudinal Microscopy of the Mouse Ovary.

The ovary is a dynamic organ that undergoes dramatic remodeling throughout the ovulatory cycle. Maturation of the ovarian follicle, release of the oocyte in the course of ovulation as well as formation and degradation of corpus luteum involve tightly controlled remodeling of the extracellular matrix and vasculature. Ovarian tumors, regardless of their tissue of origin, dynamically interact with the ovarian microenvironment. Their activity in the tissue encompasses recruitment of host stroma and immune cells, attachment of tumor cells to mesothelial layer, degradation of the extracellular matrix and tumor cell migration. High-resolution dynamic imaging of such processes is particularly challenging for internal organs. The implementation of a novel imaging window as reported here enabled longitudinal microscopy of ovarian physiology and orthotopic tumor invasion.

Equine chorionic gonadotropin alters luteal cell morphologic features related to progesterone synthesis.

Exogenous eCG for stimulation of a single dominant follicle or for superovulation are common strategies to improve reproductive efficiency by increasing pregnancy rates and embryo production, respectively. Morphofunctional changes in the CL of eCG-treated cattle include increases in CL volume and plasma progesterone concentrations. Therefore, we tested the hypothesis that eCG alters the content of luteal cells and mitochondria related to hormone production. Twelve crossbred beef cows were synchronized and then allocated into three groups (four cows per group) and received no further treatment (control) or were given eCG either before or after follicular deviation (superovulation and stimulation of the dominant follicle, respectively). Six days after ovulation, cows were slaughtered and CL collected for morphohistologic and ultrastructural analysis. Mitochondrial volume per CL was highest in superovulated followed by stimulated and then control cows (18,500 ± 2630, 12,300 ± 2640, and 7670 ± 3400 µm(3); P < 0.001), and the density of spherical mitochondria and the total number of large luteal cells were increased (P < 0.05) in stimulated cows compared with the other two groups (110.32 ± 14.22, 72.26 ± 8.77, and 70.46 ± 9.58 mitochondria per µm(3) and 678 ± 147, 245 ± 199, and 346 ± 38 × 10(6) cells, respectively. However, the largest diameters of the large luteal cells were increased in superovulated and control cows versus stimulated ones (32.32 ± 0.06, 31.59 ± 0.81, and 29.44 ± 0.77 µm; P < 0.0001). In contrast, the total number of small luteal cells was increased in superovulated cows (1456 ± 268, 492 ± 181, and 822 ± 461 × 10(6), P < 0.05). In conclusion, there were indications of cellular changes related to increased hormonal production (stimulatory treatment) and increased CL volume (superovulatory treatment).

Topographic distribution of the different cell types, connective tissue and vascular tissue/lumina within a functional bovine corpus luteum and its association with breed, type of fixation protocol and stage during the cycle.

In the present study, we analysed the effect of fixative, breed, luteal stage and location on the nuclear density, volume density of connective tissue and vascular tissue/lumina within a bovine luteal gland in view of the development of an in vivo sampling technique to longitudinally monitor luteal histophysiology. The inner zone defined as the zone geometrically closest to the centre of the gland shows a significantly lower nuclear density (for all cell types) and a higher volume density of collagen fibres and vessels when compared with the outer zone (p < 0.001). The nuclear density in luteal glands from Holstein-Friesian cows is not significantly different from that in Belgian Blue cows, nor is it in stage II vs stage III glands. The collagen fibre content was significantly lower in glands of Belgian Blue cows (p = 0.01) and in younger glands (p = 0.003). Hence, it seems that the lower nuclear density in the inner zone was compensated by a higher amount of collagen fibres. As the type of fixative applied has a significant effect on the nuclear density of the different cell types, the present study warrants future research to further optimize the fixation protocol. As a conclusion, we can state that the topographic difference in nuclear distribution for the different cell types in a bovine luteal gland is only significant when comparing the inner vs the outer zone. This implies that if a sample representative for the whole gland has to be taken, for example, when taking an in vivo sample, it is necessary that the biopsy goes through the inner zone and contains the total diameter of the gland.

Restoration of corpus luteum angiogenesis in immature hypothyroid rdw rats after thyroxine treatment: morphologic and molecular evidence.

Thyroxine (T4) plus gonadotropins might stimulate ovarian follicular angiogenesis in immature infertile hypothyroid rdw rats by upregulating mRNA expression of major angiogenic factors. Development of growing corpus luteum (CL) is strongly related to angiogenesis and to morphofunctional development of microcirculation. Our aim was to investigate if T4 is involved in CL angiogenesis and in the activation of capillary cells and angiogenic factors after ovulation in a spontaneous model of hypothyroidism, the rdw rat. Rdw rats were treated with T4 plus gonadotropins (equine chorionic gonadotropin plus human chorionic gonadotropin; eCG+hCG) or gonadotropins alone in order to evaluate the effects of T4 on early luteal angiogenesis, on microvascular cells and on expression of major growth factors which are involved in the regulation of angiogenesis. Wistar-Imamichi rats treated with gonadotropins were used as controls. The ovaries were collected 4 days after hCG administration and analyzed using morphologic and molecular approaches. Thyroxine plus gonadotropins stimulated the growth of CLs and follicles as in controls, differently from rdw rats treated only with gonadotropins, in which CLs were not found and only small follicles, often atretic, could be recognized. In T4 plus gonadotropin-treated rdw rats CLs showed increased microvasculature, numerous activated capillaries characterized by sprouting and other angiogenic figures, and associated pericytes. Quantitative analysis revealed that the number of pericytes in T4 plus gonadotropin-treated rdw rats was comparable with that found in control rats and was significantly higher than that found in gonadotropin-treated rdw rats. The mRNA expression of vascular endothelial growth factor and basic fibroblast growth factor was significantly higher in control rats and in T4 plus gonadotropin-treated rdw rats than in gonadotropin-treated rdw rats. mRNA expression of tumor necrosis factor α, transforming growth factor ß, and epidermal growth factor did not show significant changes. Our data originally demonstrated that T4 promoted the growth of an active microcirculation in developing CLs of gonadotropin-primed hypothyroid rdw rats, mainly by inducing sprouting angiogenesis, pericyte recruitment, and upregulation of mRNA expression of vascular endothelial growth factor and basic fibroblast growth factor. In conclusion, we suggest that T4 plays a key role in restoring luteal angiogenesis in ovaries of immature hypothyroid rdw rats.

Differential morphological effects in rat corpora lutea among ethylene glycol monomethyl ether, atrazine, and bromocriptine.

Ethylene glycol monomethyl ether (EGME) or atrazine induces luteal cell hypertrophy in rats. Our previous study suggested that EGME stimulates both new and old corpora lutea (CL), while atrazine stimulates new CL. Bromocriptine (BRC) is known to suppress the luteolysis in rats. This study investigated the light- and electron-microscopic luteal changes induced by EGME, atrazine, or BRC. Female rats were treated with EGME (300 mg/kg/day), BRC (2 mg/kg/day), EGME and BRC (EGME + BRC), or atrazine (300 mg/kg/day) for 7 days. Luteal cell hypertrophy induced by EGME, EGME + BRC, and atrazine was subclassified into the following two types: CL hypertrophy, vacuolated type (CL-V) characterized by intracytoplasmic fine vacuoles, and CL hypertrophy, eosinophilic type (CL-E) characterized by eosinophilic and abundant cytoplasm. The proportions of CL-V and CL-E were different among the treatments. BRC-treated old CL showed lower proportion of endothelial cells and fibroblasts than normal old CL. Ultrastructural observation revealed that the luteal cells of CL-V contained abundant lipid droplets, whereas those of CL-E in EGME and EGME + BRC groups showed uniformly well-developed smooth endoplasmic reticulum. No clear ultrastructural difference was observed between the control CL and atrazine-treated CL-E. These results indicate that EGME, atrazine, and BRC have differential luteal morphological effects.

Allopregnanolone alters the luteinizing hormone, prolactin, and progesterone serum levels interfering with the regression and apoptosis in rat corpus luteum.

Steroids synthesized in the central nervous system are termed "neurosteroids". They are synthesized and metabolized in several brain areas. The objective of this work was to determine if 1 intracerebroventricular allopregnanolone injection in rats can interfere in luteal regression in a close relationship with modifications in LH, progesterone, and prolactin serum concentrations. Allopregnanolone was injected during proestrus morning and the animals were sacrificed on oestrous morning. Ovulation test and histological analysis were performed in the oestrus morning with light and electron microscopy. Serum prolactin, LH, and progesterone levels were measured by radioimmunoassay. The allopregnanolone injection significantly decreased luteinizing hormone serum level and the number of oocytes on oestrus. Progesterone and prolactin serum levels were increased after this injection. The inhibition of apoptotic figures due to allopregnanolone administration was detected in the already formed corpora lutea belonging to the previous ovary cycle and it was significantly lower than in vehicle group (control). When the GABA(A) antagonist (bicuculline) was administered alone or previously to allopregnanolone, no effect on the ovulation rate was observed. No changes in the apoptotic cell numbers were observed with respect to those of vehicle group. These results show that the effect of centrally injected allopreganolone over reproductive function could be due to a centrally originated LH mediated effect over ovarian function that affects luteal regression, through the inhibition of apoptosis and stimulation of progesterone and prolactin release.

Ultrasound image analysis offers the opportunity to predict plasma progesterone concentrations in the estrous cycle in cows: a feasibility study.

In recent years, several attempts have been made to evaluate the activity of a corpus luteum by determining its sonographic echo texture. In all of these studies the values of the echo texture parameters depended on the type and settings of the ultrasound machine. Therefore, the aim of the study was to investigate if a quantitative analysis of ultrasound (US) images of the corpus luteum (CL) after calibration of the ultrasound machine enables the assessment of the peripheral plasma progesterone (P4) level. Ten Holstein Friesian cows were examined daily at Days 4 to 8, 10 to 16, and -5 to -1 (Day 1=ovulation) of the estrous cycle. B-mode sonography of the corpora lutea was performed and blood samples were taken for plasma P4 analysis. US images were calibrated and analyzed using a software package (CAUS) developed by the authors. In addition to the area of the CL (Total Area, TotA; Tissue Area interactive, TisAi; Tissue Area Automatic, TisAa), the following US parameters were calculated from the gray level histogram and from the size of the speckles: Mean, Standard Deviation (SD) and Signal-to-Noise Ratio (SNR=Mean/SD) of echo levels, Residual Attenuation (ResAtt), Axial and Lateral speckle size (Ax and Lat, respectively). The inter-individual variability of the P4 level was expressed by the coefficient of variability (CV), averaged over all days. It appeared that the CV of the absolute P4 was high (0.65) and the P4 relative to that at Day 4 and at Day 16 was of comparable magnitude. Correlations of US parameters with P4 were highest for the P4 relative to Day 16 (P4_rel_D16). This relative P4 measure was then used for further analysis. The correlations of P4_rel_D16 with TotA, TisAa (CL area after automatic segmentation of tissue) and ResAtt were found the highest (R=0.68, 0.74, and -0.42, respectively). Multiple linear regression analysis, incorporating all US parameters revealed the formula: P4_rel_D16(pred)=-0.315+0.225TisAa-0.023ResAtt, and a goodness of fit: R(2)=0.59 (p<0.001). This formula was then used to "predict" for each image the P4_rel_D16 from the estimated US parameters. A high correlation of the predicted with the measured P4_rel_D16 was found: R=0.77. Classification of images using the predicted P4_rel_D16 to be in the range >0.80 (corresponding to 0.95 times the average_P4_rel_D16 measured during the "static" phase of the luteal cycle) by ROC analysis was correctly made in 88% of cases. In conclusion the quantitative analysis of calibrated ultrasound images may yield a good prediction of cyclic changes of P4 levels and has potential for predicting the phase in the estrous cycle of a cow.

Efectos de la selección y de la superovulación en las pérdidas gestacionales de ratonas CF1 / Effects of selection and superovulation on gestational losses in CF1 mice

En un par de líneas de ratones seleccionadas para alto (s') y bajo peso (s), originadas a partir de una población no seleccionada de la cepa CF1 (t), se modificó la estructura ovárica. El diámetro de los folículos ováricos y el número de folículos y de cuerpos lúteos se incrementaron en las hembras de la línea s', sin expresarse en un mayor tamaño de camada al nacimiento, posiblemente, por un aumento de las pérdidas gestacionales. Se probó si los efectos conjuntos de la selección de peso a largo plazo y de la estimulación ovárica incrementaban las pérdidas gestacionales. Se utilizaron dos grupos de hembras por línea: sin y con estimulación ovárica (5UI de eCG y 5UI de hCG). Las hembras se sacrificaron a las 56-72 hs y a los 7 días postservicio y después de la primera parición. Se observaron los números de cuerpos lúteos (CL), embriones (E) y sitios de implantación (SI) y el tamaño de camada al nacimiento (TC). Se estimaron las pérdidas totales (PT) y las pérdidas de cuerpos lúteos (PCL), de embriones (PE) y de fetos (PF). Los promedios de CL, E, SI y TC variaron en el mismo sentido de la selección practicada y fueron significativamente mayores (P<0,05) para las hembras estimuladas, a excepción de TC. La línea s' tuvo un potencial reproductivo superior pero un mayor costo biológico (mayor PT y más tardía) cuando se la comparó con las otras líneas. La estimulación ovárica produjo menores eficiencias reproductivas totales para las tres líneas y pérdidas gestacionales mayores y más tardías, principalmente de SI. Las hembras de la línea no seleccionada (t), no estimuladas, con pesos intermedios, parieron un mayor número de crías, partiendo de un número intermedio de CL, E y SI, con una menor y más temprana mortalidad embrionaria, demostrando ser las más eficientes desde el punto de vista reproductivo y productivo.

The ovarian structure was modified as a consequence of weight selection in a pair of mouse lines selected for high (s') and low weight (s). Lines were founded from an unselected population of CF1 strain (t). The follicle diameter and the number of the ovarian follicles and the corpora lutea were higher in s' females, but they did not reach a larger litter size at birth, may be, by an increase in the gestational losses. In these lines, the co-effects of long-term weight selection and ovarian stimulation were tested to evaluate if they increased gestational losses. Two groups of females per line were employed: without and with ovarian stimulation (5UI of eCG and 5UI of hCG). Females were slaughtered at 56-72hs and at 7 days post-breeding and after first parturition. The number of corpora lutea (CL), embryos (E) and implantation sites (SI), and litter size at birth (TC) were observed. Total losses (PT) and corpora lutea (PCL), embryo (PE) and fetus (PF) losses were estimated. Mean CL, E, SI and TC varied in the same direction of the selection made and they were significantly higher (P<0.05) in stimulated females, though not for TC. Line s' had a higher reproductive potential but a greater biological cost (higher and later gestational mortality) when compared with the other lines. Ovarian stimulation produced lower total reproductive efficiencies for the three lines and higher and later gestational losses, mainly for implantation sites. Females from unselected line (t), without ovarian stimulation, with intermediate weights, bore larger litters, starting from an intermediate number of CL, E and SI, with a lower and earlier embryo mortality, showing to be the most efficient from a reproductive and productive point of view.

Role of the DLL4-NOTCH system in PGF2alpha-induced luteolysis in the pregnant rat.

We investigated the expression and cell localization of NOTCH1, NOTCH4, and the delta-like ligand DLL4 in corpus luteum (CL) from pregnant rats during prostaglandin F2alpha (PGF2alpha)-induced luteolysis. We also examined serum progesterone (P(4)) and CL proteins related to apoptosis after local administration of the notch inhibitor N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT). Specific staining for NOTCH1 and NOTCH4 receptors was detected predominantly in large and small luteal cells. Furthermore, in line with the fact that the notch intracellular domain is translocated to the nucleus, where it regulates gene expression, staining was evident in the nuclei of luteal cells. In addition, we detected diffuse cytoplasmic immunostaining for DLL4 in small and large luteal cells, in accordance with the fact that DLL4 undergoes proteolytic degradation after receptor binding. The mRNA expression of Notch1, Notch4, and Dll4 in CL isolated on Day 19 of pregnancy decreased significantly after administration of PGF2alpha. Consistent with the mRNA results, administration of PGF2alpha to pregnant rats on Day 19 of pregnancy decreased the protein fragment corresponding to the cleaved forms of NOTCH1/4 CL receptors. In contrast, no significant changes were detected in protein levels for the ligand DLL4. The local intrabursal administration of DAPT decreased serum P(4) levels and increased luteal levels of active caspase 3 and the BAX:BCL2 ratio 24 h after the treatment. These results support a luteotropic role for notch signaling to promote luteal cell viability and steroidogenesis, and they suggest that the luteolytic hormone PGF2alpha may act in part by reducing the expression of some notch system members.

Local effects of the sphingosine 1-phosphate on prostaglandin F2alpha-induced luteolysis in the pregnant rat.

Since the regression of the corpus luteum (CL) occurs via a tightly controlled apoptotic process, studies were designed to determine if local administration of the antiapoptotic agent sphingosine 1-phosphate (S1P) effectively blocks the luteolytic action of prostaglandin F-2alpha (PGF-2alpha). On day 19 of pregnancy, 2 hr before systemic PGF-2alpha administration, rats were injected intrabursa with either S1P or vehicle (control). The activity of four caspases, which contribute to the initial (caspase-2, -8, and -9) and final (caspase-3) events in apoptosis was measured in pooled CL from four individual ovaries at 0 and 4 hr after PGF-2alpha injection. The expression of the phosphorylated form of AKT (pAKT) and tumor necrosis factor-alpha (TNF-alpha) was analyzed by ELISA. In addition, cell death was evaluated by electronic microscopy (EM) in CL 4 and 36 hr after PGF-2alpha injection. The activity of caspase-2, -3, and -8 was significantly greater by 4 hr after PGF-2alpha, but not caspase-9 activity. In contrast, expression of pAKT and TNF-alpha decreased significantly. Administration of S1P suppressed (P < 0.05) these effects, decreasing caspase activities and increasing pAKT and TNF-alpha expression. The administration of S1P also significantly decreased the percentage of luteal apoptotic cells induced by PGF-2alpha. PGF-2alpha treatment increased the prevalence of luteal cells with advanced signs of apoptosis (i.e., multiple nuclear fragments, chromatin condensation, or apoptotic bodies). S1P treatment suppressed these changes and increased the blood vessel density. These results suggest that S1P blocks the luteolytic effect of the PGF-2alpha by decreasing caspase-2, -3, and -8 activities and increasing AKT phosphorylation and TNF-alpha expression.

Population of follicles and luteal structures during the oestrous cycle of mares detected by three-dimensional internal structure microscopy.

The structure of the equine ovary is different from that of other mammals in its extremely large size, the presence of ovarian fossa and the inverted location of its cortex and medulla. A three-dimensional internal structure microscopy (3D-ISM), which consists of a computer-controlled slicer, a CCD camera, a laser disc recorder and a PC, is very useful for the observation of the internal structures in equine ovaries. In addition, the three-dimensional images of follicles and corpus luteum (CL) reconstructed by the segmentation technique can clarify the spatial arrangement in the equine ovary. In this study, to understand the changes in the ovarian internal structures of the mare during the oestrous cycle, the size and numbers of follicles and luteal structures were analysed by 3D-ISM in addition to the concentrations of progesterone (P(4)) and oestradiol-17beta. As a result, many small follicles (<10 mm in diameter) were detected. It was recognized that the luteal structures were distinguished into three types, such as the corpus haemorragicum (CH), which is formed by blood elements at the cavity after ovulation, CL and corpus albican (CA). There were some CHs and CL in the group, which had the concentration of P(4) > 1 ng/ml. CHs were also observed in the group, which had low level of P(4) (P(4) < 1 ng/ml). CAs were found regardless of the P(4) level. In conclusion, 3D-ISM enabled the internal observation of the ovarian structures in detail, and estimation of the stage of the ovarian cycle with complementary physiological information. The findings by 3D-ISM provide basic information for clinical applications.

Features of natural and gonadotropin-releasing hormone antagonist-induced corpus luteum regression and effects of in vivo human chorionic gonadotropin.

CONTEXT: The natural process of luteolysis and luteal regression is induced by withdrawal of gonadotropin support. OBJECTIVE: The objectives of this study were: 1) to compare the functional changes and apoptotic features of natural human luteal regression and induced luteal regression; 2) to define the ultrastructural characteristics of the corpus luteum at the time of natural luteal regression and induced luteal regression; and 3) to examine the effect of human chorionic gonadotropin (hCG) on the steroidogenic response and apoptotic markers within the regressing corpus luteum. DESIGN: Twenty-three women with normal menstrual cycles undergoing tubal ligation donated corpus luteum at specific stages in the luteal phase. Some women received a GnRH antagonist prior to collection of corpus luteum, others received an injection of hCG with or without prior treatment with a GnRH antagonist. MAIN OUTCOME MEASURE: Main outcome measures were plasma hormone levels and analysis of excised luteal tissue for markers of apoptosis, histology, and ultrastructure. RESULTS: The progesterone and estradiol levels, corpus luteum DNA, and protein contents in induced luteal regression resembled those of natural luteal regression. hCG treatment raised progesterone and estradiol in both natural luteal regression and induced luteal regression. The increase in apoptosis detected in induced luteal regression by cytochrome c in the cytosol, activated caspase-3, and nuclear DNA fragmentation, was similar to that observed in natural luteal regression. The antiapoptotic protein Bcl-2 was significantly lower during natural luteal regression. The proapoptotic proteins Bax and Bak were at a constant level. Apoptotic and nonapoptotic death of luteal cells was observed in natural luteal regression and induced luteal regression at the ultrastructural level. hCG prevented apoptotic cell death, but not autophagy. CONCLUSION: The low number of apoptotic cells disclosed and the frequent autophagocytic suggest that multiple mechanisms are involved in cell death at luteal regression. hCG restores steroidogenic function and restrains the apoptotic process, but not autophagy.

Immunohistochemical localization of androgen receptor and aromatase in the ovary of the pregnant pig.

The distribution of androgen receptor (AR) and cytochrome P450 aromatase was investigated in paraffin sections of pregnant pig ovary. Ovarian follicles and corpora lutea were isolated from ovaries obtained on Days 10, 18, 32, 71 and 90 post coitum (p.c.). Androgen receptor was localized in the nuclei of granulosa cells of follicles of various sizes. In addition, some follicles demonstrated staining in the nuclei of theca interna cells. Stroma cells also exhibited a positive immunostaining. At early and mid pregnancy (up to Day 71) AR was expressed in the nuclei of luteal cells. Corpora lutea of Day 71 showed mainly cytoplasmic staining while on Day 90 almost all luteal cells showed staining exclusively in the cytoplasm. Immuno-staining for the presence of cytochrome P450 aromatase was very faint in all investigated ovarian structures. The results could suggest that the process of androgen aromatization plays a negligible role in the ovary of the pregnant pig.

The effects of superior ovarian nerve sectioning on ovulation in the guinea pig.

The effects on spontaneous ovulation associated with the unilateral or bilateral sectioning of the superior ovarian nerves (SON) were analyzed in guinea pigs at different time intervals of the estrous cycle. Day 1 of the estrous cycle was defined as the day when the animal presents complete loss of the vaginal membrane (open vagina). Subsequent phases of the cycle were determined by counting the days after Day 1. All animals were autopsied on the fifth day of the estrous cycle after surgery. Sectioning the right, left, or both SONs on day 5 (early luteal phase) resulted in a significant increase in the number of fresh corpora lutea. Ovulation increased significantly when the left SON (L-SON) was sectioned during late follicular phase (day 1) and medium luteal phase (day 8). When surgery was performed on days 1 or 8, neither sectioning the right SON (R-SON) nor sectioning the SON bilaterally had an apparent effect on ovulation rates. Similarly, ovulation rates were not affected when unilateral (right or left) or bilateral sectioning of the SON was performed during late luteal phase two (day 12). Unilateral or bilateral sectioning of the SON performed during the early luteal phase (day 5) was associated with a significant decrease in uterine weight. A comparable effect was observed when the L-SON was sectioned during late follicular phase (day 1), or medium luteal phase (day 8). No effects on uterine weight were observed when unilateral or bilateral sectioning of the SON was performed during late luteal phase. Our results suggest that in the guinea pig the SON modulates ovulation, and that the degree of modulation varies along the estrous cycle. The strongest influence of the SONs on ovulation occurs during early luteal phase, and decrease thereafter, being absent by late luteal phase. In addition, sectioning the left or the right SON caused different responses by the ovaries of adult guinea pigs. This paper discusses the mechanisms by which ovulation increased when the SON was surgically cut.

Cell death during natural and induced luteal regression in mares.

In mares, little information is available on the type of cell death that occurs during natural and induced luteal regression. Corpora lutea were collected from mares in the early luteal phase, days 3-4 (n = 4); mid-luteal phase, day 10 (n = 5); early regression, day 14 (n = 4); late regression, day 17 (n = 4); and 12 and 36 h (n = 3 per group) after PGF2alpha administration on day 10. Histological and ultrastructural sections were examined and TUNEL was used to detect DNA fragmentation. In early luteal regression, there were more pyknotic luteal cells and extracellular round dense bodies compared with the mid-luteal phase. By late regression, there was a significant decline (P < 0.01) in the number of round dense body clusters and a marked accumulation of lipid. Twelve and 36 h after PGF2alpha administration, changes were similar to those seen in natural regression, but there was also a marked infiltration of neutrophils. Accumulation of lipid was not apparent until 36 h after PGF2alpha administration. Ultrastructural examination revealed rarefaction and distortion of the mitochondrial cristae in most of the luteal cells by the mid-luteal phase. Luteal cells showed shrinkage, accumulation of lipid with foamy appearance, and disruption in both smooth endoplasmic reticulum and mitochondria during natural and induced regression. Some luteal cells showed fragmented or pyknotic chromatin characteristic of apoptosis. Other luteal cells showed crenation of the nuclear membrane and shrinkage of the nucleus, features not characteristic of apoptotic cell death. In late regression, capillaries were obstructed by swollen endothelial cells and round dense bodies. These results show that structural regression may be initiated as early as the mid-luteal phase, and is clearly visible by day 14 in natural regression and 12 h after induced regression. Apoptosis did appear to be involved in luteolysis in the equine corpus luteum, but non-apoptotic changes were also observed in some luteal cells during regression. Accumulation of lipid was a late feature of luteal regression.

Apoptosis induced by antigestagen RU486 in rat corpus luteum of pregnancy.

Administration of RU486 to late pregnant rats results in preterm delivery 24 h after treatment and the induction of a luteolytic process after labor. We investigated whether functional changes occurring within the corpora lutea after RU486 treatment were associated with morphologic features of apoptotic cell death. Rats on d 18 of pregnancy were treated with RU486 (5 mg/kg) at 10:00 am and killed 72 h after. We studied the number of apoptotic cells in paraffin sections of the corpora lutea by routine hematoxylin and eosin (H&E) staining, and by in situ 3' end labeling (TdT-mediated dUTP nick-end labeling [TUNEL]). The corpora lutea were also processed for electron microscopy to study ultrastructural changes after RU486 treatment. The number of cells showing apoptotic nuclei in H&E-stained sections was higher in RU486-treated animals than in controls (vehicle-treated rats). The quantification of the number of apoptotic nuclei within the corpora lutea performed by TUNEL confirmed the higher number of apoptotic nuclei in animals receiving the antigestagen compared with controls. Ultrastructurally, the luteal cells undergoing apoptosis presented a highly deteriorated cytoplasmic organization The nuclei, in an initial step of regression, displayed condensation of the chromatin, a prominent nucleolus, and a perinuclear space. In an advanced step of degeneration, the nuclei showed evidence of large irregular aggregates of condensed chromatin. Prostaglandin F2,alpha(PGF2alpha), which mediates the luteolytic action of RU486, mimicked the effect of the antigestagen on the induction of apoptosis when administered to rats on d 18 of pregnancy (100 microg at 9:00 am and 1:00 pm), which were killed 72 after the last injection. In conclusion, the present results indicate that functional luteolysis in rats is associated with structural luteal regression with the morphologic features of apoptotic cell death, as demonstrated by studying the luteolytic process induced by the administration of the antigestagen RU486.

The fate of corpora lutea in the cyclic golden hamster.

Structural luteolysis shows striking interspecies differences. Morphological changes in the corpus luteum (CL) of the cyclic hamster have been studied alongside the potential involvement of known luteolytic hormones. Ovaries from intact Syrian golden hamsters killed at 1100 h on days 1 and 2 and at 1100 and 1700 h on days 3 and 4 of the estrous cycle were dissected for histological study. The day of ovulation, the day of estrus, was arbitrarily designated day 1 of the estrous cycle. Steroidogenic cells in the CL were scarcely luteinized on day 1 and reached full luteinization on day 2. On the morning of day 3, initial regressive changes (accumulation of lipid droplets, invasion by neutrophils, and accumulation of phagocytic cells) were observed. These regressive changes increased progressively and apoptotic cells as well as phagocytic cells containing phagocytized apoptotic cells were abundant on the evening of day 3. On the morning of day 4, apoptotic cells/bodies and phagocytic cells containing phagocytized material were extremely abundant throughout the CL. However, steroidogenic cells with intact nuclei and well-preserved blood vessels were also found. Surviving cells in the CL showed progressive morphological changes. These cells showed morphological features intermediate between luteal and interstitial cells in the evening of day 4 and were virtually indistinguishable from interstitial cells on day 1 of the following cycle. Additional animals were injected at 1100 h on day 2 with: (a) the dopaminergic agonist CB154 (0.4 mg) to block prolactin secretion, (b) the anti-estrogen LY117018 (1.6 mg) or the anti-androgen Flutamide (3 mg) to block estrogen or androgen receptors, respectively, and (c) progesterone (2 mg) to prevent the fall in serum progesterone concentrations. Ovaries from these animals were collected at 1700 h on day 3 and at 1000 h on day 4. The luteolytic process was not affected by any treatment. These data indicate that, in contrast to its close relatives (e.g., the rat), structural luteolysis in the hamster is independent of the apoptotic inducing luteolytic hormones. In addition, differences in the cellular mechanisms responsible for CL elimination were also present. In the hamster, part of the luteal cells do not undergo apoptosis and seemed to progress through another developmental path giving rise to interstitial-like cells.

Effect of dose of prostaglandin F(2alpha) on steroidogenic components and oligonucleosomes in ovine luteal tissue.

To determine whether prostaglandin (PG) F(2alpha) had a dose-dependent effect upon secretion of progesterone, oligonucleosome formation, or loss of luteal weight, ewes on Day 9 or 10 of the estrous cycle were administered 0, 3, 10, or 30 mg PGF(2alpha) per 60 kg BW (i.v.), and luteal tissue was collected 9 and 24 h after injection. All doses of PGF(2alpha) decreased (P < 0. 05) concentrations of progesterone in sera by 9 h; however, in ewes treated with 3 mg PGF(2alpha), concentrations of progesterone were similar to control values at 24 h and higher (P < 0.05) than those in the 10- or 30-mg groups. Concentrations of progesterone in sera over all dose levels were highly correlated to luteal concentrations of mRNA encoding steroidogenic acute regulatory protein (P < 0.001), cytochrome P450 side-chain cleavage (P < 0.02), and 3beta-hydroxysteroid dehydrogenase (P < 0.01). Corpora lutea collected at 24 h from ewes treated with the 10- and 30-mg doses of PGF(2alpha) weighed less (P < 0.05) than those from controls. Oligonucleosomes were not present in luteal tissues from control ewes. Surprisingly, all doses of PGF(2alpha)-induced oligonucleosomes in a majority of animals at 9 h and in a majority of ewes treated with 10 and 30 mg of PGF(2alpha) at 24 h. In conclusion, 3 mg of PGF(2alpha) per 60 kg BW transiently decreased serum concentrations of progesterone and induced oligonucleosome formation, but did not result in reduced luteal weight. The 10- and 30-mg doses of PGF(2alpha) decreased secretion of progesterone and induced oligonucleosome formation and luteolysis.