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.     

Luteoprotective roles of luteinizing hormone are mediated by not only progesterone production but also glucocorticoid conversion in bovine corpus luteum

Luteinizing hormone (LH) is known as a key regulator of corpus luteum (CL) function, but the luteoprotective mechanisms of LH in the maintenance of bovine CL function are not well understood. The current study investigated if LH increases cell viability and induces cortisol conversion, and if the luteoprotective action of LH is mediated by stimulating the local production and action of progesterone (P4) and/or cortisol. Cultured bovine luteal cells obtained at the mid‐luteal stage (Days 8–12 of the estrous cycle) were treated for 24 hr with LH (10 ng/ml) with/without onapristone (OP, a specific P4 receptor antagonist; 100 µM), cortisone (1 µM), and aminoglutethimide (AGT, a specific inhibitor of cytochrome P450 side‐chain cleavage; 100 µM). LH with and without OP significantly increased the mRNA and protein expressions of 11β‐hydroxysteroid dehydrogenase (HSD11B) 1, but did not affect the mRNA or protein expression of HSD11B2. These treatments also significantly increased HSD11B1 activity. Cell viability was significantly increased by LH alone or by LH in combination with cortisone and OP. LH in combination with OP or AGT significantly decreased cell viability as compared to LH alone. The overall results suggest that LH stimulates not only P4 production but also HSD11B1 expression, thereby increasing the cortisol concentration in the bovine CL, and that LH prevents cell death through these survival pathways. LH may consequently support CL function during the luteal phase in cattle. Mol. Reprod. Dev. 80: 204–211, 2013. © 2013 Wiley Periodicals, Inc.     

Expression of LH receptor mRNA splice variants in bovine granulosa cells: changes with follicle size and regulation by FSH in vitro

In cattle, most evidence suggests that granulosa cells express LH receptors (LHR) after (or as) the follicle becomes dominant, however there is some suggestion that granulosa cells from smaller pre-dominant follicles may express several LHR mRNA splice variants. The objective of this study was to measure LHR expression in bovine follicles of defined size and steroidogenic ability, and in granulosa cells from small follicles (<6 mm diameter) undergoing differentiation in vitro. Semiquantitative RT-PCR demonstrated that LHR mRNA was undetectable in granulosa cells of follicles <7 mm diameter (nondominant follicles), and increased with follicle diameter in follicles >7 mm diameter. Splice variants with deletions of exon 10 and part of exon 11 were detected as previously described, and we detected a novel splice variant with a deletion of exon 3. Cultured granulosa cells contained LHR mRNA, but with significantly greater amounts of variants with deletions of exon 10 and/or exon 11 compared with cells from dominant follicles. FSH increased the abundance of some but not all LHR mRNA splice variants in cultured granulosa cells. The addition of LH to cultured cells did not increase progesterone secretion, despite the presence of LHR mRNA. Collectively, these data suggest that granulosa cells do not acquire functional LHR until follicle dominance occurs.     

Expression of monocyte chemoattractant protein-1 and distribution of immune cell populations in the bovine corpus luteum throughout the estrous cycle.

This study characterizes the expression of monocyte chemoattractant protein-1 (MCP-1) and the relative distribution of immune cell populations in the bovine corpus luteum throughout the estrous cycle. Immunodetectable MCP-1 was evident in corpora lutea of cows at Days 6, 12, and 18 postovulation (Day 0 = ovulation, n = 4 cows/stage). Day 6 corpora lutea contained minimal MCP-1 that was confined primarily to blood vessels. In contrast, relatively intense staining for MCP-1 was observed in corpora lutea from Days 12 and 18 postovulation. MCP-1 was again most evident in the cells of the vasculature, but it was also observed surrounding individual luteal cells, particularly by Day 18. An increase in immunohistochemical expression of MCP-1 on Days 12 and 18 postovulation corresponded with increases in MCP-1 mRNA and protein in corpora lutea as determined by Northern blot analysis and ELISA. Monocytes and macrophages were the most abundant immune cells detected in the bovine corpus luteum, followed by CD8+ and CD4+ T lymphocytes. In all instances, Day 6 corpora lutea contained fewer immune cells than corpora lutea from Days 12 and 18. In conclusion, increased expression of MCP-1 was accompanied by the accumulation of immune cells in the corpora lutea of cows during the latter half of the estrous cycle (Days 12-18 postovulation). These results support the hypothesis that MCP-1 promotes immune cell recruitment into the corpus luteum to facilitate luteal regression. These results also raise a provocative issue, however, concerning the recruitment of immune cells several days in advance of the onset of luteal regression.     

Tissue concentration, mRNA expression and stimulation of IGF-I in luteal tissue during the oestrous cycle and pregnancy of cows

The expression of IGF-I in bovine luteal tissue was demonstrated by parallel measurement of IGF-I tissue concentration and its mRNA; highest synthesis was observed during Days 12-17 of the cycle and the first months of pregnancy. Tissue levels of IGF-I increased from Days 1-5 to Days 12-17 of the cycle followed by a rapid decrease at luteolysis; there was a continuous decline from early pregnancy until Months 6-9. Microdialysis perfusion experiments with corpora lutea in vitro at Days 8-11 of the cycle revealed a major effect: release of progesterone and oxytocin were highly stimulated in a dose-dependent manner. We suggest that IGF-I could be important in regulating the function of the bovine corpus luteum and may act in an autocrine/paracrine way.     

Expression of mRNA encoding insulin-like growth factors I and II and the type 1 IGF receptor in the bovine corpus luteum at defined stages of the oestrous cycle

Previous studies have implicated insulin-like growth factors I and II (IGF-I and -II), in the regulation of ovarian function. The present study investigated the localization of mRNA encoding IGF-I and -II and the type 1 IGF receptor using in situ hybridization to determine further the roles of the IGFs within the bovine corpus luteum at precise stages of the oestrous cycle. Luteal expression of mRNA encoding IGF-I and -II and the type 1 IGF receptor was detected throughout the oestrous cycle. The expression of IGF-I mRNAvaried significantly during the oestrous cycle. IGF-I mRNA concentrations were significantly higher on day 15 than on day 10, and IGF-I mRNA in the regressing corpus luteum at 48 h after administration of exogenous prostaglandin was significantly greater than in the early or mid-luteal phase (days 5 and 10). In contrast, there was no significant effect of day of the oestrous cycle on expression of mRNA for IGF-II and the type 1 IGF receptor in the corpus luteum. Expression of IGF-II mRNA was localized to a subset of steroidogenic luteal cells and was also associated with cells of the luteal vasculature. mRNA encoding the type 1 IGF receptor was widely expressed in a pattern indicative of expression in large and small luteal cells. These data demonstrate that the bovine corpus luteum is a site of IGF production and reception throughout the luteal phase. Furthermore, this study highlights the potential of IGF-II in addition to IGF-I in the autocrine and paracrine regulation of luteal function.     

Oestradiol administration raises luteal LH receptor levels in intact and hysterectomized pigs

Occupied and unoccupied LH receptors in corpora lutea, and LH and progesterone concentrations in circulating plasma, were measured in non-pregnant gilts that had been treated with oestradiol-17 beta benzoate to prolong luteal function. Oestradiol benzoate (5 mg, administered on Day 12 after oestrus) delayed luteal regression and the decline in LH receptor levels at luteolysis and raised unoccupied receptor levels from 11.8 +/- 1.14 fmol/mg protein on Days 10--15 after oestrus to 31.8 +/- 3.26 fmol/mg protein on Days 15--21. There was no simultaneous rise in occupied receptor levels and occupancy decreased from 29.8 +/- 3.01 to 11.5 +/- 1.26%. Basal plasma LH concentrations were unchanged by oestradiol, but mean corpus luteum weight and plasma progesterone concentrations were slightly reduced. Oestradiol benzoate on Day 12 caused a similar increase in unoccupied receptor levels in gilts hysterectomized on Days 6--9 after oestrus, from 17.0 +/- 5.83 to 34.5 +/- 6.00 fmol/mg protein, determined on Days 15--18. Plasma concentrations of LH and progesterone were unchanged by oestradiol. Unoccupied receptor levels in corpora lutea and plasma LH and progesterone were unaltered by hysterectomy in untreated gilts. Occupied receptor levels were not influenced by hysterectomy or oestradiol. It is concluded that oestradiol-17 beta raises luteal LH receptor levels by a mechanism independent of the uterus.     

Differential patterns in luteal prolactin and LH receptors during pregnancy in sows and ewes

In the sow, a dramatic increase of LH specific binding was observed during the second half of pregnancy. This was due to an increase in receptor number (41 fmol and 95 fmol/mg protein at Days 50 and 105 respectively). The apparent association constant was unchanged. The pattern of prolactin receptor content showed two peaks at Day 60 and Day 105. Prolactin receptors increased earlier during pregnancy than did LH receptors, suggesting a possible role of prolactin in the induction of LH receptors. In the ewe, the receptor content of LH and prolactin did not change very much during pregnancy. The corpus luteum showed normal luteal function until parturition although it was not necessary for maintenance of pregnancy in the ewes.     

Bovine membrane-type 1 matrix metalloproteinase: molecular cloning and expression in the corpus luteum

Matrix metalloproteinase-2 (MMP-2) is produced as a zymogen, which is subsequently activated by membrane-type 1 metalloproteinase (MT1-MMP). The objectives of the present study were to clone bovine MT1-MMP and to investigate its expression in the corpus luteum. Corpora lutea were harvested from nonlactating dairy cows on Days 4, 10, and 16 of the estrous cycle (Day 0 = estrus; n = 3 for each age). The bovine MT1-MMP cDNA contained an open reading frame of 1749 base pairs, which encoded a predicted protein of 582 amino acids. Northern blotting revealed no differences (P > 0.05) in MT1-MMP mRNA levels between any ages of corpora lutea. Western blotting demonstrated that two species of MT1-MMP, the latent form ( approximately 63 kDa) and the active form ( approximately 60 kDa), were present in corpora lutea throughout the estrous cycle. Active MT1-MMP was lower (P < 0.05) in early stages of the corpus luteum than the mid and late stages, where MMP-2 activity, as revealed by gelatin zymography, was also elevated. Furthermore, immunohistochemistry revealed that MT1-MMP was localized in endothelial, large luteal, and fibroblast cells of the corpus luteum at different stages. Taken together, the differential expression and localization of MT1-MMP in the corpus luteum suggest that it may have multiple functions throughout the course of the estrous cycle, including activation of pro-MMP-2.     

Expression of mRNA for follicle-stimulating hormone suppressing protein in ovarian tissues of cows.

The expression of bovine follicle-stimulating hormone (FSH)-suppressing protein (FSP) mRNA was investigated in different ovarian tissues of cows. Northern blot analysis, using a cDNA probe to bovine FSP, demonstrated that the FSP gene in the bovine ovary is highly expressed in a pool of isolated granulosa cells. Two bands (2.8 and 1.8 kb) were observed in all tissues expressing the mRNA. FSP mRNA was low in small antral follicles and increased in growing follicles to reach a maximum in preovulatory follicles. Low amounts of mRNA of steady state FSP were observed in all stages of the corpus luteum as well as in the corpus luteum of pregnant cows, in the corpus albicans and theca tissue, whereas this mRNA could not be detected in the liver. These results are consistent with the hypothesis that, in cows, FSP functions as an autocrine regulator in developing follicles to facilitate luteinization of granulosa cells.     

Identification and physical mapping of genes expressed in the corpus luteum in cattle

A representational difference analysis was performed to identify genes expressed in the corpus luteum of cattle. The corpus luteum is an ovarian structure that is essential for the establishment and maintenance of pregnancy. Knowledge of gene expression and function of corpus luteum will be important to improve fertility in humans and domestic animals. Housekeeping genes were removed from the corpus luteum representation (tester) using skeletal muscle as the subtracting agent (driver). A total of 80 clones of the final subtraction product were analysed by sequencing and 11 new bovine gene sequences were identified (pBTCL1-11). The sequences were mapped to segments of 10 different chromosomes using a somatic cell hybrid panel and a radiation hybrid panel. With one exception the locations are in agreement with published comparative maps of cattle and man. Expression in corpus luteum was verified by RT-PCR for all the 11 clones.     

Growth hormone, but not luteinizing hormone, acts with luteal peptides on prostaglandin F2alpha and progesterone secretion by bovine corpora lutea in vitro*

Prostaglandin F2alpha (PGF2alpha) is a major physiological luteolysin in the cow. However, injection of PGF2alpha before day 5 (day 0 = estrus) of the estrous cycle dose not induce luteolysis. On the other hand, the early corpus luteum (CL) actively produces PGF2alpha. This indicates that luteal PGF2alpha may play a key role in the refractoriness to PGF2alpha injected during the early luteal phase when angiogenesis is active in the CL. Thus, this study aimed to investigate the possible interaction between pituitary hormones and local factors (luteal peptides) on secretion of PGF2alpha and progesterone (P) by the early bovine CL, and to evaluate the effect of growth hormone (GH) as well as its interactions on production of PGF2alpha in the developing CL. A RT-PCR analysis revealed that mRNA for GH receptor in CL was fully expressed from early in the luteal phase throughout the estrous cycle, while luteinizing hormone (LH) receptor mRNA was expressed less by the early and regressing CL than those at mid or late luteal phases (P < 0.05). For the stimulation test, an in vitro microdialysis system (MDS) was used as a model. Each bovine early CL (days 3-4) was implanted with the MDS, and maintained in an organ culture chamber. The infusion of GH, insulin-like growth factor-1 (IGF-1) and oxytocin (OT) increased (P < 0.05) PGF2alpha and P release. In contrast, LH had no effect (P > 0.05) on PGF2alpha secretion and little effect on P release. Unexpectedly, there was no distinct interaction between pituitary hormones and luteal peptides on secretion of PGF2alpha and P. These results indicate that GH is a more powerful stimulator of PGF2alpha and P production in the early bovine CL than LH and suggest that GH and luteal peptides, IGF-1 and OT, contribute to maintenance of elevated PGF2alpha production in the developing bovine CL.     

Growth hormone, but not luteinizing hormone, acts with luteal peptides on prostaglandin F2alpha and progesterone secretion by bovine corpora lutea in vitro

Prostaglandin F2alpha (PGF2alpha) is a major physiological luteolysin in the cow. However, injection of PGF2alpha before day 5 (day 0 = estrus) of the estrous cycle dose not induce luteolysis. On the other hand, the early corpus luteum (CL) actively produces PGF2alpha. This indicates that luteal PGF2alpha may play a key role in the refractoriness to PGF2alpha injected during the early luteal phase when angiogenesis is active in the CL. Thus, this study aimed to investigate the possible interaction between pituitary hormones and local factors (luteal peptides) on secretion of PGF2alpha and progesterone (P) by the early bovine CL, and to evaluate the effect of growth hormone (GH) as well as its interactions on production of PGF2alpha in the developing CL. A RT-PCR analysis revealed that mRNA for GH receptor in CL was fully expressed from early in the luteal phase throughout the estrous cycle, while luteinizing hormone (LH) receptor mRNA was expressed less by the early and regressing CL than those at mid or late luteal phases (P < 0.05). For the stimulation test, an in vitro microdialysis system (MDS) was used as a model. Each bovine early CL (days 3-4) was implanted with the MDS, and maintained in an organ culture chamber. The infusion of GH, insulin-like growth factor-I (IGF-I) and oxytocin (OT) increased (P < 0.05) PGF2alpha and P release. In contrast, LH had no effect (P > 0.05) on PGF2alpha secretion and little effect on P release. Unexpectedly, there was no distinct interaction between pituitary hormones and luteal peptides on secretion of PGF2alpha and P. These results indicate that GH is a more powerful stimulator of PGF2alpha and P production in the early bovine CL than LH and suggest that GH and luteal peptides, IGF-1 and OT, contribute to maintenance of elevated PGF2alpha production in the developing bovine CL.     

Regulation and role of the insulin-like growth factor I system in rat luteal cells.

The relationship between insulin-like growth factor I (IGF-I), a hormone which has potent metabolic effects and stimulates protein synthesis, and prolactin and oestradiol was examined to investigate a possible mechanism for the luteal cell hypertrophy that is responsible for the increase in size of the corpus luteum. A luteal cell line (GG-CL) derived from large luteal cells of the pregnant rat corpus luteum was used. IGF-I, IGF-I receptor and oestrogen receptor beta mRNA contents were determined by semiquantitative RT-PCR. The results revealed that prolactin upregulates the expression of IGF-I mRNA in luteal cells, but not that of its receptor. IGF-I had no effect on the expression of its receptor but caused a dose-related increase in the expression of oestrogen receptor beta. Furthermore, whereas IGF-I upregulated oestrogen receptor beta expression, oestradiol downregulated expression of mRNA for both IGF-I and its receptor. This effect of oestradiol is not mediated through progesterone which is stimulated by oestradiol in the corpus luteum. The developmental studies indicate that mRNA for IGF-I and its receptor are not expressed in tandem throughout pregnancy. Whereas the receptor mRNA is expressed at higher concentrations in early pregnancy, that of its ligand is highly expressed close to parturition. Collectively, the results indicate that prolactin stimulates luteal IGF-I production, which in turn acts on the luteal cell to stimulate expression of oestrogen receptor beta. Luteal cells with increased oestrogen receptor beta can respond fully to oestradiol, leading to cell hypertrophy.     

Hammond memorial lecture. New concepts of the control of corpus luteum function

Five new concepts concerning the control of corpus luteum function in the cow have been developed in recent years. Prostacyclin (PGI-2) plays a luteotrophic role. Conversely, products of the lipoxygenase pathway of arachidonic acid metabolism, particularly 5 hydroxyeicosatetraenoic acid (5-HETE), play luteolytic roles. Luteal cells arise from two sources. The small luteal cells are all of theca cell origin; the large cells found early in the cycle (Days 2-6) are mainly of granulosa cell origin. However, a population of large cells found after Day 10 of the cycle are of theca cell origin. Oxytocin of luteal cell origin plays a role in development of the corpus luteum and possibly in its regression. The recently described Ca2+-polyphosphoinositol-protein kinase C second messenger system, as well as the LH-cAMP system, is involved in control of progesterone synthesis in the bovine corpus luteum. Progesterone synthesis in the small theca-derived luteal cells is primarily controlled by the cAMP system. However, elevated intracellular calcium diminishes cAMP-mediated progesterone synthesis in these cells. These findings modify our current concepts of the mechanisms of control of progesterone secretion by the corpus luteum and suggest several new lines of research.     

FSH receptors in the bovine corpus luteum

Binding of follicle stimulating hormone (FSH) to a crude membrane fraction of bovine corpus luteum (CL) has been detected. This binding meets the usual criteria for a receptor based on specificity, time course of reaction and association constant (Ka = 8.5 x 10(10)M(-1)). Physiological studies with CL removed from heifers at specific times after estrus indicate that day-6 CL had the highest FSH binding. However, a correlation with physiological function was not obvious since some functional mid-cycle CL were high in progesterone and luteinizing hormone (LH) receptor but had nondetectable FSH receptor. Conversely, some late-cycle CL had low progesterone and LH receptor but significant quantities of FSH receptor.