A histological study of corpora lutea from superovulated beef heifers

There is great variability between animals in the number of viable embryos produced following different superovulation regimens. It is not clear if all the follicles that ovulate produce healthy oocytes and form normal corpora lutea (CL) following superovulation. The objective of this study was to assess and compare CL from heifers undergoing three superovulatory regimes with CL from unstimulated heifers on the basis of morphology and morphometric analysis of luteal cells.Beef heifers were superovulated using either: (a) 24 mg porcine follicle stimulating hormone (pFSH) given twice daily over a 4 day period in decreasing doses commencing on day 10 of the oestrous cycle; (b) a single injection of 2000 IU pregnant mare serum gonadotrophin (PMSG) given on day 10 of the cycle; (c) as in (b) but followed by 2000 IU anti-PMSG (IgG to neutralise endogenous PMSG) at the time of the first insemination which was 12–18 h after the onset of oestrus (n = 33 per treatment). Luteolysis was induced 48 h after initial gonadotrophin administration and CL were collected on day 7 of the subsequent cycle and from ten unstimulated heifers (controls) at the same stage of the oestrous cycle. CL morphology was studied at light and electron microscopy levels. Morphometric analysis was performed on luteal cells. Subcellular morphology was similar in heifers from all groups. However, CL from superovulated heifers had more connective tissue than CL from control heifers; the connective tissue content of CL in the anti-PMSG-treated group was particularly marked. Both large and small luteal cells in the heifers receiving anti-PMSG had significantly smaller (P < 0.001) area and sphere volume than similar cells from CL of heifers in the other groups.     

Stimulation of seasonally anovular merino ewes by rams. II. Premature regression of ram-induced corpora lutea

Ovulation was induced by rams in 74 of 91 seasonally anovular Merino ewes. The resulting corpora lutea (CL) were observed by laparoscopy and were found to either persist normally ($\text{38}\text{74}$), or regress prematurely ($\text{36}\text{74}$). In 32 of the latter ewes premature regression of the CL was followed by a second ovulation within 6 days of the introduction of rams.     

Prostaglandin-induced regression of porcine corpora lutea maintained by estrogen

Corpora lutea were marked with suture in 24 crossbred gilts on day 7 to 9 of the estrous cycle (first day of estrus = 0). All gilts were injected with 5 mg of estradiol benzoate (EB) daily from day 10 to 15 to extend the lifespan of corpora lutea, then the gilts were randomly assigned to two groups. On day 20, the 12 gilts of Group 1 were injected with 10 mg PGF_2α, and the 12 gilts of Group 2 were injected with saline. Ovaries were recovered 10 to 13 days after PGF_2α or saline injection. Ten gilts in Group 1 displayed estrus 5 ± 0.7 days after PGF_2α injection, but only two gilts in Group 2 displayed estrus during the experimental period. In gilts that displayed estrus, all marked CL had regressed. Marked CL were still present in all 12 gilts that failed to exhibit estrus during the experimental period. These results show that in the pig, PGF_2α caused regression of CL that were maintained beyond the normal luteal phase of the estrous cycle by EB treatment.     

Plasma progesterone levels in superovulated cattle in relation to hatched embryo sizes,ovulation rates,and premature regression of corpora lutea

Progesterone concentrations were measured in peripheral plasma of 62 cattle treated with PMSG. There was good correlation (r = 0.92) between a single day-10 value and the sum of daily values up to day 12 (19 animals) or day 13 (17 animals). The day-10 progesterone level was correlated strongly with ovulation rate (r = 0.76; 55 animals) but to a negligible extent with the sizes of 306 day-13 embryos from 47 donors (r = 0.17). Premature regression of corpora lutea, encountered in 14 (7.3%) of 191 flushed donors, began between days 5 and 8 and was reflected in the progesterone profiles of seven animals that were serially sampled.     

Luteinizing hormone,progesterone and the morphological development of normal and superovulated corpora lutea in sheep

The development of granulosa-lutein cells was studied in 27 normal and 32 superovulated ewes between days 0-4(day 0 began with the preovulatory LH peak in normal animals and the HCG injection in superovulated ewes). The pattern of differentiation was similar in both groups. Following initial hormonal stimulation (0-12 hours after LH or HCG), granulosa cells were approximately 100 mu2 and contained small, pleomorphic nuclei with large amounts of clumped chromatin. Elongate cells lining the basement membrane possessed large, heterogeneous dense bodies, and a well-developed Golgi apparatus. Mitotic figures were observed up to 6 hours prior to ovulation. Sixteen to 20 hours following the LH surge or HCG injection, hypertrophy of granulosa cells was evident. Nuclei contained definitive nucleoli. Blood vessels in the theca interna were abundant and highly dilated. Ovulation occurred approximately 24 hours after the LH peak or HCG injection. Visible signs of luteinization were evident 6-12 hours after ovulation. A slight increase in serum progesterone levels was detected. The second post-ovulatory day was characterized by continuing hypertrophy of granulosa cells and extensive proliferation of smooth endoplasmic reticulum and mitochondria. Nuclei of granulosa cells were larger and possessed extremely large nucleoli. Numerous mitotic figures were apparent within the corpus luteum. Serum progesterone concentrations began increasing at 60-72 hours after hormone stimulation. By the end of the third post-ovulatory day, the corpus luteum consisted of large, pleomorphic, parenchymal cells, interspersed between capillaries and connective tissue elements. Only an occasional mitotic figure was apparent within the corpus luteum at 100 hours. Light microscopic autoradiography of 5, 10, and 15 day corpora lutea taken from ewes pulsed with 3H thymidine at specific times before and after ovulation revealed that granulosa cells did not undergo secondary mitoses following ovulation. In contrast, thecal, mesenchymal and endothelial cells did mitose on day 3.     

Prostaglandin-induced regression of porcine corpora lutea maintained by estrogen.

Corpora lutea were marked with suture in 24 crossbred gilts on day 7 to 9 of the estrous cycle (first day of estrus equals 0). All gilts were injected with 5 mg of estradiol benzoate (EB) daily from day 10 to 15 to extend the lifespan of corpora tutea, then the gilts were randomly assigned to two groups. On day 20, the 12 gilts of Group 1 were injected with 10 mg PGF-2ALPHA, and the 12 gilts of Group 2 were injected with saline. Ovaries were recovered 10 to 13 days after PGF-2ALPHA or saline injection. Ten gilts in Group 1 displayed estrus 5 plus or minus 0.7 days after PGF-2ALPHA injection, but only two gilts in Group 2 displayed estrus during the experimental period. In gilts that displayed estrus, all marked CL had regressed. Marked CL were still present in all 12 gilts that failed to exhibit estrus during the experimental period. These results show that in the pig, PGF-2ALPHA caused regression of CL that were maintained beyond the normal luteal phase of the estrous cycel by EB treatment.     

Development of preovulatory follicles expected to form short-lived corpora lutea in beef cows

Oestrus, expected to be followed by a short luteal phase, was induced in post-partum cows by weaning their calves at 35 days after parturition. Ovaries containing the first preovulatory follicles (Type F) formed after parturition were collected 3 h after the onset of oestrus. For comparison, preovulatory follicles (Type C) were collected 3 h after the onset of oestrus in normally cycling cows. The number of granulosa cells was determined and the concentrations of receptors for follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in granulosa cells and for LH in theca cells were measured. Concentrations of oestradiol-17 beta, testosterone, androstenedione and progesterone in follicular fluid were also measured. Type F follicles contained about twice the number of granulosa cells (based on DNA) as did Type C follicles (45.8 +/- 11.3 and 24.5 +/- 3.9 micrograms DNA/follicle, respectively; P less than 0.05) but these cells had fewer receptors for LH (0.13 +/- 0.02 vs 0.29 +/- 0.03 fmol/micrograms DNA; P less than 0.01) and FSH (0.61 +/- 0.08 vs 1.3 +/- 0.29 fmol/micrograms DNA; P less than 0.08) than did those from Type C follicles. Additionally, there were fewer receptors for LH in theca tissue from Type F than from Type C follicles (28.3 +/- 5.2 vs 51.3 +/- 6.1 fmol/follicle; P less than 0.01). Concentrations of oestradiol-17 beta (475.8 +/- 85.6 vs 112.9 +/- 40.0 ng/ml; P less than 0.01) and androstenedione (214.1 +/- 48.7 vs 24.7 +/- 7.7 ng/ml; P less than 0.01) in follicular fluid were higher in Type C than in Type F follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Premature ovulations in superovulated cattle

The incidence and consequences of premature ovulations in superovulated cattle were studied. These ovulations, presumably induced by the luteinizing hormone content of the gonadotrophin preparation used, were found in 17 of 133 (13%) stimulated cows and heifers used as oocyte donors. In general this group of donors exhibited deviating periovulatory patterns of plasma hormone profiles (progesterone, estradiol17beta and LH), estrus behaviour, follicular steroidogenesis of progesterone and estradiol-17beta, and oocyte meiosis. It is concluded that superovulated donor cattle exhibiting premature ovulations constitute a group of poor oocyte donors that also must be considered as inferior embryo donors.     

Formation and regression of capillary sprouts in corpora lutea of immature superstimulated golden hamsters

The ultrastructure of developing and regressing capillary sprouts was studied in corpora lutea of immature golden hamsters between days 4 and 7 after the application of serum gonadotrophin of pregnant mares (PMSG). Horseradish peroxidase (HRP), an endothelial tracer, was localized by ultrahistochemistry. The vascular permeability of HRP was quantified by an enzyme assay in ovarian homogenates. Sprouting endothelial cells looked activated. They showed micropinocytotic vesicles in a high endothelium surrounded by basal laminae. Early capillary growth was at its maximum on day 4 after PMSG. Advanced capillary growth was seen on days 4 and 5 after PMSG. The vascular lumina were formed by dilatation of the interendothelial space. Regression of capillary sprouts started on day 5, was most intense on day 6 and negligible on day 7. Two processes of regression were observed. One led to a complete destruction, the other to an incomplete one. Vascular permeability decreased between days 5 and 6 after PMSG. It is concluded that the corpus luteum can be viewed as a physiological model of angiogenesis.     

Mitogenic factors of corpora lutea

The mammalian corpus luteum (CL), which plays a central role in the reproductive process because of its production of hormones such as progesterone, appears to be an exceptionally dynamic organ. Its rate of growth and development are extremely rapid and, even when the CL is functionally mature, its rate of cell turnover remains relatively high. Associated with this high rate of cell turnover, the mature CL receives the greatest blood supply per unit tissue of any organ, and also exhibits a relatively high metabolic rate. Although numerous growth factors have been identified in luteal tissue, their role in growth and differentiation of this dynamic organ remains unclear. Recently, while attempting to identify mitogenic factors of ovine and bovine CL, we have found that they produce several mitogens during the estrous cycle as well as pregnancy. The majority of these luteal-derived mitogenic factors are heparin-binding, and although some may represent previously identified factors, several appear to be novel heparin-binding growth factors. Isolation and purification of mitogenic factors produced by the CL will enable us to determine their roles in luteal growth, development and differentiated function, which will contribute to our understanding not only of the regulation of fertility but also of tissue growth and development in general.     

Cytokeratin expression in bovine corpora lutea

Cytokeratin (CK)-positive cells were obtained from bovine corpora lutea. When cultured, these cells behave like CK-positive endothelial cells obtained from bovine large blood vessels. The origin of CK-positive cells has now been studied in 45 bovine corpora lutea of different estrous cycle stages. Additionally, 7 corpora lutea of pregnant cows were examined. The tissues were grouped into early stage (days 2 to 4), secretory stage (days 5 to 17) and late stage (days 18 to 21) according to gross morphology, wet weight and total progesterone content. One portion of a corpus luteum was used for immunohistochemistry, and another for Western blot analysis. Twenty-six of the 45 corpora lutea showed CK expression, as confirmed by immunostaining and Western blotting. Cytokeratin expression was found in all corporalutea from the early stage, in 14 of 26 corpora lutea from the secretory stage, and 3 of 10 from the late stage. Early stage corpora lutea displayed zonation such that a high number of CK-positive luteal cells occurred in the region of the previous granulosa layer and a very low number in the previous thecal layer. Secretory CK-positive corpora lutea showed uniformly distributed, predominantly large luteal cells. In secretory corpora lutea of group A, CK-positive cells and a distinct microvascular tree were seen, the latter visualized by factor VIII-related antigen immunolabelling of endothelial cells. Group B showed none or very few CK-positive cells. Corpora lutea of pregnant cows behaved like corpora lutea of group B. Roughly 1% of CK-positive cells closely associated with the capillary wall were sometimes reminiscent of endothelial cell sprouts.     

In vivo oxytocin release from microdialyzed bovine corpora lutea during spontaneous and prostaglandin-induced regression

The release of luteal oxytocin during spontaneous and prostaglandin-induced luteolysis was investigated in cows. A continuous-flow microdialysis system was used in 11 cows to collect dialysates of the luteal extracellular space between Days 12 and 24 postestrus. Seven cows were untreated and were expected to exhibit spontaneous luteolysis during sampling, whereas 4 cows received prostaglandin F(2alpha) (PGF(2alpha)) systemically between Days 13 and 15 to induce luteolysis during sampling. Oxytocin was detectable in the dialysate of all cows before Day 16 postestrus and occurred as 2 or 3 discrete pulses per 12-h sampling period. For non-PGF(2alpha)-treated cows, dialysate oxytocin content began to decline spontaneously on Day 15 postestrus and was undetectable by Day 17 postestrus. Oxytocin decay curves preceded onset of serum progesterone decline by at least 72 h and were not related temporally with onset of progesterone decline within cow. Exogenous PGF(2alpha) (25 mg, i.m.) produced a 10-fold increase in dialysate oxytocin within 1 h (1.9 +/- 0.3 pg/ml to 20.8 +/- 3.0 pg/ml; P < 0. 01). Dialysate oxytocin then declined to pretreatment concentrations within 2 h and was undetectable within 8 h posttreatment. A second PGF(2alpha) injection given 20 h after the first did not result in a measurable increase in dialysate oxytocin, probably because luteolysis was underway. Although robust luteal oxytocin release was observed after treatment with a pharmacological dose of PGF(2alpha), the lack of detectable oxytocin secretion during spontaneous luteolysis suggests that the contribution of luteal oxytocin in the cow may be less than that proposed for the ewe.     

Failure of human chorionic gonadotropin injections to sustain gonadotropin-releasing hormone-induced corpora lutea in postpartum beef cows

Anestrous postpartum (PP) Hereford cows (n =20) were used to determine the effects of repeated injections of human chorionic gonadotropin (hCG) on the progesterone (P4) secretion and functional lifespan of gonadotropin-releasing hormone (GnRH)-induced corpora lutea (CL). Suckling was reduced to once a day from Day 21 to Day 25 PP, and all cows received injections of 200 micrograms GnRH at 1500 h on Day 24 PP to induce ovulation. Treated cows (HCG, n = 10) received 200 IU hCG b.i.d. from 1900 h on Day 27 PP to 1900 h on Day 33 PP; control cows (CTRL, n=10) were not injected. Blood was collected on Days 21, 23, 25, and 27 to 33, 35, 37, and 39 PP. Serum P4 concentration was measured by radioimmunoassay and used to classify luteal lifespan and the associated estrous cycle as short (SHORT) or normal (NORM) in duration. Treatment with hCG resulted in more (p less than 0.01) cows with SHORT cycles (7 of 9 vs. 4 of 9). Serum P4 concentrations were similar (p greater than 0.20) between groups from 4 days before until 6 days after GnRH injection. Cows with NORM cycles (n = 7) had greater serum P4 concentrations (p less than 0.05) on Days 7 to 11 after GnRH than cows with SHORT cycles (n = 11). By Day 39 PP, all cows with SHORT cycles appeared to have undergone a second ovulation. Charcoal-stripped serum pools from before (PRE) and during hCG injection (INJ) were assayed for total luteinizing hormone-like bioactivity (LH-BA) using a dispersed mouse-Leydig cell bioassay.(ABSTRACT TRUNCATED AT 250 WORDS)     

GnRH-like proteins in cows: concentrations during corpora lutea development and selective localization in granulosal cells

Gonadotropin-releasing hormone (GnRH)-like proteins with anti-gonadotropic properties were recently discovered in the ovaries of several species, including humans. Since neither GnRH receptors nor GnRH are in bovine ovarian tissue, we examined, in the present studies, whether concentrations of GnRH-like proteins varied during development of the corpus luteum (CL) and whether GnRH-like proteins were selectively localized in ovarian cells of cows. For these studies, GnRH-like proteins were extracted from various ovarian and nonovarian tissues and fluids and fractionated for hydrophobic interaction chromatography. A highly specific and sensitive radioreceptor assay (RRA) was used to quantify concentrations of GnRH-like proteins. The major findings of these studies demonstrated that 1) the amount of GnRH-like proteins in the corpus luteum (CL) was proportional to the weight of the CL; 2) the concentration of GnRH-like proteins in luteal tissue decreased during development of the CL; 3) GnRH-like proteins were in ovarian and numerous nonovarian tissues, but were not in the heart, plasma, or follicular fluid; 4) the retention time for GnRH-like proteins following high-pressure liquid chromatography (HPLC) varied with the tissue source; and 5) compared with all other tissues, the greatest concentration of GnRH-like proteins was in granulosal cells. We concluded that the concentration of GnRH-like proteins in luteal cells decreased during development of the CL, and that a specific GnRH-like protein was selectively localized in bovine granulosal cells.     

Luteal function of induced corpora lutea in the bitch

Nineteen anestrous bitches with a mean of 22 kg body weight and ranging from 2 to 4 years of age were induced to exhibit estrus and ovulate using PMSG and HCG. Twelve days after the first day of estrus, bitches were assigned to four treatment groups. Group (A) consisted of six bitches, Group (B) of five bitches and Groups (C) and (D) of four bitches each. At this time, bitches in Groups (A), (B) and (C) were laparotomized and those assigned to Groups (A) and (B) were bilaterally hysterectomized leaving the cervix and oviducts intact. Although bitches in Group (C) were laparotomized, they were not hysterectomized. Group (D) bitches were not subjected to any surgical procedures. Homologous uterine extract was prepared from each bitch in Group (A) and administered intramuscularly beginning on day 25 (day 0 = first day of estrus) and continued every other day for 61 days post-estrus. Bitches in Group (B) were similarly injected with equal volumes of 0.9% saline. Blood samples, obtained prior to laparotomy and every other day for 85 days thereafter, were assayed for plasma progesterone concentrations using radioimmunoassay. One bitch in each of Groups (A) and (D) did not form luteal tissue following treatment with PMSG and HCG although both bitches exhibited estrus following treatment. All other bitches showed an increase in progesterone levels (4 to 19 ng/ml) between the first day of estrus and 10 days post-estrus. Thereafter, progesterone levels progressively declined in all groups with levels below 1 ng/ml between 38 to 40 days post-estrus. Results of this study suggested that CL formed in the bitch following PMSG and HCG treatment have a reduced function compared to non-induced CL of a normal, non-fertile estrous cycle. Such premature CL regression appears to be independent of the presence or absence of the uterus.     

Function of hormonally-induced corpora lutea in the domestic cat

Hysterectomy (3 animals) or diversion of the uterine venous blood from the ovarian vasculature (3 animals) has no effect on the duration of hormonally-induced luteal activity in the pseudopregnant luteal-phase cat (control: 4 animals), suggesting that the uterus exerts no local or systemic influence on the duration of luteal function.