Time related changes in luteal prostaglandin synthesis and steroidogenic capacity during pregnancy, normal and antiprogestin induced luteolysis in the bitch

In nonpregnant and pregnant dogs the corpora lutea (CL) are the only source of progesterone (P4) which shows an almost identical secretion pattern until the rapid decrease of P4 prior to parturition. For the nonpregnant dog clear evidence has been obtained that physiological luteal regression is devoid of a functional role of the PGF2α-system and seems to depend on the provision of StAR. Yet in pregnant dogs the rapid prepartal luteal regression, coinciding with an increase of PGF2α, may be indicative for different regulatory mechanisms. To assess this situation and by applying semi-quantitative Real Time (Taq Man) RT-PCR, expression patterns were determined for the following factors in CL of pregnant and prepartal dogs and of mid-pregnant dogs treated with the antiprogestin Aglepristone: cyclooxygenase 2 (Cox2), prostaglandin E2 synthase (PGES), prostaglandin F2α synthase (PGFS), its receptors (EP2, EP4 an FP), the steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid-dehydrogenase (3βHSD) and the progesterone receptor (PR). Peripheral plasma P4 concentrations were determined by RIA. CL were collected via ovariohysterectomy from pregnant bitches (n = 3–5) on days 8–12 (Group 1, pre-implantation period), days 18–25 (Group 2, post-implantation period), days 35–40 (Group 3, mid-gestation period) and during the prepartal progesterone decline (Group 4). Additionally, CL were obtained from groups of 5 mid-pregnant dogs (days 40–45) 24 h, respectively 72 h after the second treatment with Aglepristone. Expression of Cox2 and PGES was highest during the pre-implantation period, that of PGFS and FP during the post-implantation period. EP4 and EP2 revealed a constant expression pattern throughout pregnancy with a prepartal upregulation of EP2. 3βHSD and StAR decreased significantly from the pre-implatation period to prepartal luteolysis, it was matched by the course of P4 concentrations. Expression of the PR was higher during mid-gestation and prepartal luteolysis than in the two preceding periods. After application of Aglepristone the overall mRNA-expression resembled the situation during prepartal luteolysis except for EP2, which remained unchanged.These data suggest that – as in the nonpregnant bitch – also in the pregnant bitch luteal production of prostaglandins is associated with luteal support rather than luteolysis. On the other hand induction of luteolysis by the PR blocker Aglepristone points to a role of luteal P4 as an autocrine factor in a positive loop feedback system controlling the availability of P4, StAR and 3βHSD.     

Plasma concentrations of 13,14-dihydro-15-keto prostaglandin F2-alpha (PGFM), progesterone and estradiol in pregnant and nonpregnant diestrus cross-bred bitches

The canine corpus luteum (CL) typically sustains elevated plasma progesterone concentrations for 2 months or more, with a peak approximately 15-25 days after ovulation, followed by a slow decline. The processes involved in the slow, protracted regression of the CL over the remaining 1.5-2-month period in nonpregnant bitches and until shortly prepartum in pregnant bitches are not well characterized. The rapid luteolysis that occurs immediately prepartum appears to be a result of a prepartum rise in peripheral PGF. The potential role of PGF in the slow regression process in the several weeks preceding parturition and in nonpregnant bitches after 15-25 days after ovulation is not known. Therefore, plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F2-alpha (PGFM), progesterone (P4) and estradiol (E2) were determined and compared in bitches during nonpregnant diestrus (n = 9) or pregnancy (n = 8). During the gradual decrease in plasma concentrations of progesterone in both groups, the P4 pattern appeared unrelated to changes in either E2 or PGFM concentrations. The PGFM pattern was different between diestrus and pregnant bitches (P > 0.01); there was an apparent progressive but slow increase in PGFM in pregnant bitches from Days 30 to 60, followed by a large increase prior to parturition; concentrations declined immediately postpartum. However, there were no increases in PGFM during the same interval in nonpregnant bitches. Mean estradiol concentrations were sporadically elevated during the last third of pregnancy and less so in nonpregnant diestrus; there was no acute prepartum increase in estradiol associated with the PGFM increase. In summary, although there were no apparent changes in peripheral PGF2alpha concentration involved in regulating the slow protracted phase of luteal regression in nonpregnant bitches, modest increases in PGFM may play a role in ovarian function after mid-gestation in pregnant bitches. Furthermore, the acute prepartum rise in PGFM was not dependent on any concomitant increase in estradiol concentrations.     

Loss of luteal sensitivity to luteinizing hormone underlies luteolysis in cattle: A hypothesis

By virtue of the secretion of progesterone (P4), corpus luteum (CL) is important not only for normal cyclicity but also for conception and continuation of pregnancy in female mammals. Luteolysis (also called luteal regression) is defined as loss of the capacity to synthesize and secrete P4 followed by the demise of the CL. There is strong evidence that sequential pulses of prostaglandin F2α (PGF) secreted from the uterus near the end of luteal phase induces luteolysis in farm animals. Loss of luteal sensitivity to luteinizing hormone (LH) at the end of menstrual cycle has been reported to be critical for initiation of luteolysis in primates, however this has not been investigated in farm animals. A closer observation of the published real-time profiles of circulating hormones (P4, LH, and PGF) and their inter-relationships around the time of the beginning of spontaneous luteolysis in cattle revealed- 1) A natural pulse of PGF causes a transient P4 suppression lasting a couple of hours followed by a rebound in P4 concentration, 2) The P4 secretions that occur in response to LH pulses before the beginning of luteolysis (i.e., preluteolysis) either fail or do so to a lesser extent during luteolysis indicating a loss of sensitivity to LH, and 3) The loss of sensitivity coincides with the beginning of luteolysis (i.e., transition), and apparently luteolysis does not initiate until there is loss of sensitivity to LH. The CL is sensitive to LH during preluteolysis, and the LH-stimulated P4-dependent and/or independent local survival mechanisms maintain the steroidogenic capability and viability of the CL until the very end of preluteolysis. Luteolysis does not appear to initiate with the PGF pulse(s) that occur during this period. With the loss of sensitivity to LH at the transition, however, a progressive decline in P4 begins initiating luteolysis. Also, the survival mechanisms become compromised making the CL less viable. The uterine PGF pulses that occur after the beginning of luteolysis induces increase in the local luteolytic factors, which contribute to further luteolysis, more importantly, structural luteolysis with ultimate demise of the CL. Therefore, I hypothesize that the loss of luteal sensitivity to LH underlies luteolysis in cattle. The hypothesis not only unifies the basic mechanism of luteolysis in a farm animal and primates but also provides a perspective to view luteolysis as a process rather than a factor-mediated event. A novel unified working model for luteolysis in a farm animal and primates is described. A better understanding of the luteal physiology including how responsiveness to LH diminishes in aging CL would help in the development of novel strategies in modulating CL structure-function to improve and/or control fertility in humans as well as in animals.     

In vitro PGF2alpha production by endometrium and corpus luteum explants from pregnant and nonpregnant diestrus bitches and placental explants from pregnant bitches

To better understand the process of slow luteal regression of the nonpregnant cycle in dogs and the acute luteolysis that occurs prepartum, the present study investigated in vitro PGF2alpha production by the endometrium, corpus luteum and placental explants obtained at known times of the cycle from pregnant bitches (days 63, 64 and immediately postpartum; day 0 = estimated day of the ovulatory LH surge) and from nonpregnant diestrus bitches (approximately days 65, 75 and 85). Both basal PGF2alpha production and its production in the presence of the protein kinase C (PKC) stimulator 12,13-phorbol dibutyrate (PDBu) were determined. For PDBu-supplemented incubations, mean PGF2alpha production (pg/mL/mg/6 h) by endometrium explants of the nonpregnant bitches in late diestrus was highest on day 65 (205 +/- 87) and reduced to low levels (38 +/- 17 and 11 +/- 11) on days 75 and 85, respectively. The production by corpus luteum explants from these bitches was significantly less on day 65 (46 +/- 14) than that of the day 65 endometrium explants, and was slightly increased on day 85 (103 +/- 52). The corresponding mean PGF2alpha production by the endometrium explants of pregnant bitches was on average much greater (i.e., two to three-fold) compared to nonpregnant bitches (P < 0.01) and involved high concentrations at day 64 (1523 +/- 467) and postpartum, compared to somewhat lower levels on day 63 (830+/-65); luteal PGF production (165 +/- 4) was also higher than in nonpregnant bitches around day 65. For pregnant bitches, PGF production per gram of tissue in the endometrium explants was greater than for the CL or placenta explants (180 +/- 37). Therefore, the endometrium of the pregnant bitch has an increased capability to produce PGF2alpha immediately prepartum, which on a tissue weight basis, exceeds that of either corpora lutea or the placenta. However, assuming a larger mass of placental tissue in vivo, we inferred that the placenta may contribute substantially to peripheral PGF concentrations.     

Endocrinology of pregnancy in the dog: a review

Pregnancy regulation in the dog is not yet fully elucidated. Since plasma progesterone concentrations are similar in pregnant versus non-pregnant animals, it is a poor reflection on CL function and progesterone metabolism. Increased progesterone secretion by the CL in pregnant animals follows implantation and relaxin secretion by the feto-placental units. Progesterone is absolutely required to maintain pregnancy and no placental sources of progesterone have been identified. Pregnancy can be artificially maintained by progesterone administration. Prolactin secretion appears to be increased in response to the increase in relaxin production and occurs independent of estrogen production by the CL. The respective roles of LH, FSH and prolactin are still unclear, with considerable conflicting evidence among studies. However, it appears that prolactin is absolutely required, whereas LH is either permissive or facilitates CL function during pregnancy. Pre-implantation events are still poorly defined in the bitch, and no embryonic factors have been isolated or purified, preventing early pregnancy diagnosis. Parturition occurs following luteolysis, which results from the release of prostaglandin F(2alpha), which begins 36h prepartum in a process similar to that observed in other species. The role of estrogens at the time of parturition remains undefined.     

Termination of pregnancy and induction of premature luteolysis by the antiprogestagen, mifepristone, in dogs

Five pregnant beagle bitches were treated with 2.5 mg mifepristone/kg body weight, twice a day, for 4.5 days starting at Day 32 of gestation. Results of fetal ultrasonography and assay of serum progesterone concentrations every 2-4 days were compared to those in 5 control bitches. Mifepristone resulted in a premature (P less than 0.01) termination of pregnancy (36 +/- 1 vs 65 +/- 1 days), without side effects. The antiprogestagen also caused progesterone to decline to less than 1 ng/ml by Day 40-45 after the preovulatory LH peak (vs 64-67 days in controls) and reduced (P less than 0.05) mean concentrations on Days 34-50 (2.2 +/- 0.5 vs 6.3 +/- 0.3 ng/ml). The results suggest that antiprogestagen therapy is a safe means to terminate unwanted pregnancy in dogs, and that luteal function in pregnant bitches is dependent on luteotrophic support that is blocked by antiprogestagen treatment, directly or indirectly, due to termination of pregnancy.     

Immunohistochemical localization of prolactin in functioning and regressing corpus luteum of pituitary autotransplanted rats

In an attempt to shed light on the intimate mechanism by which prolactin (PRL) switches from supporting corpus luteum (CL) progesterone secretion (P) to promote structural regression of the CL, day 2 (metestrous) autopituitary transplanted (APTr) rats were used. In APTr rats the CL is under the only control of PRL since an almost complete absence of LH and FSH exist. The experimental group was given bromocriptine (CB-154: 0.4 mg/day) on days 12, 13 and 14 of the cycle and 0.25 ml of ethanol from day 15 to day 21. The control group was given CB-154 from day 12 to day 21. Rats were hemiovariectomized on day 12 to assess the morphological characteristics of the active CL. PRL and P were determined by RIA on days 12, 15 and 22. On day 12, both PRL and P levels were higher than 80 ng/ml (luteotrophic action of PRL). On day 15, due to treatment with CB-154, the levels of both hormones had fallen below 7 ng/ml (functional luteolysis). On day 22, PRL levels were again high (greater than 50 ng/ml) in the shortly CB-154-treated rats and low (less than 5 ng/ml) in the controls; the P levels were lower than 5 ng/ml in both groups. PRL-induced structural luteolysis in the experimental group (hyperprolactinemic) was assessed by the structural characteristics and by the CL weight loss on day 22 in comparison with that exhibited by control rats. The immunohistochemical staining of both endogenous and total PRL in the lutein cells showed that the internalization of PRL is not modified by the functional state of the CL, nevertheless the intracellular redistribution of the internalized hormone varied in relation with the PRL action on the CL (luteotrophic, day 12 vs luteolytic, day 22). These results seem to indicate that intracellular mechanisms rather than receptor content determine CL response to PRL.     

Reproductive cycles of the domestic bitch

Domestic dogs are monoestrous, typically non-seasonal, polytocous, spontaneous ovulators and have a spontaneous luteal phase slightly longer (by approx 5 day) than the 64±1day luteal phases of a 65±1day pregnancy, a phase followed by an obligate anestrus before the next 2-3 week "heat" (proestrus-estrus). The resulting inter-estrus intervals of 5-12 months are variable among bitches, commonly 6-7 months, and range from highly variable to regular (to perhaps within±5-10 day of sequential 7 month cycle, for instance) within bitches, and across studies and do not vary significantly between pregnant and non-pregnant cycles. Hormone levels reported are those observed in this laboratory using previously reported assays and canine gonadotropin standards unless stated otherwise. Endocrine sequences for dog cycles are not unlike those of many other mammals, including selection of ovulatory follicles by increased LH pulsatility, the occurrence of estrus behavior and LH surge during a decline in the estrogen: progestin ratio, a pronounced preovulatory luteinization as in humans and rodents, and luteotrophic roles for both LH and prolactin. Non-pregnant bitches have a spontaneously prolonged luteal phase, often longer and with a more protracted decline in serum progesterone than in pregnancy as there is no uterine luteolytic mechanism. The obligate anestrus of 8-40 weeks is terminated by poorly understood interactions of environment (e.g. pheromones, possibly photoperiod) and a potential endogenous circannual cycle in sensitivities of hypothalamic dopaminergic, serotonergic and/or opioid pathways.     

Apoptosis in canine corpus luteum during spontaneous and prostaglandin-induced luteal regression

Spontaneous luteal regression and prostaglandin-induced luteolysis in bitches were evaluated by measuring the apoptotic index for DNA fragmentation and the relative level of Bax gene expression in ovaries removed from nine untreated nonpregnant bitches at selected times during diestrus and in nine pregnant bitches after 1 day of administering abortive doses of a PGF-analog gel formulation given intravaginally at selected times during gestation. Nonpregnant diestrus was divided into three periods (early, mid and late) based on vaginal cytology and plasma progesterone concentration. Pregnant bitches were treated with a PGF-analog gel at corresponding stages of pregnancy (early, mid and late) and evaluated by ultrasound. Another eight pregnant bitches were similarly studied and serum progesterone concentrations were determined after 1, 2, 3 or 4 days of PGF-analog gel. Corpora lutea obtained by ovariohysterectomy were analyzed for apoptotic internucleosomal DNA fragmentation relative to that in a control cell line (U937), using an apoptotic DNA ladder kit and gel electrophoresis and for relative expression of the pro-apoptotic Bax gene by RT-PCR and electrophoresis. In nonpregnant bitches, the DNA fragmentation apoptotic index was greater in late than in early diestrus (P < 0.01). The index after 1 day of PGF-analog gel was higher in early pregnant bitches than in early diestrus bitches (P < 0.05); it was highest in midpregnancy (P < 0.05). The degree of apoptosis was related to the number of times PGF-analog gel was administered. Bax mRNA was detected in the corpus luteum (CL) and Bax expression increased from early to middiestrus in nonpregnant subjects (P < 0.05). Potential elevation in Bax due to PGF-analog gel treatment in pregnancy was only significant in relation to normal diestrus during early pregnancy (P < 0.01). In conclusion, we inferred that the effects of endogenous or exogenous prostaglandin on CL life span in bitches involved increases in apoptotic activity and that increased apoptosis was implicated in normal luteal regression in nonpregnant bitches.     

Canine prostaglandin F2alpha receptor (FP) and prostaglandin F2alpha synthase (PGFS): molecular cloning and expression in the corpus luteum

In the dog luteolysis is not affected by hysterectomy. This observation led to the hypothesis that paracrine/autocrine rather than endocrine mechanisms of PGF2alpha are responsible for luteal regression in the dioestric bitch. The present experiments tested for the capacity of canine CL to produce and respond to PGF2alpha by qualitatively and quantitatively determining the expressions of PGFS, the enzyme converting PGH2 into PGF2alpha, and the PGF2alpha-receptor (FP) in CL of non-pregnant dogs during dioestrus. Canine PGFS and FP were isolated and cloned; both genes show a high homology (82-94%) when compared to those of other species. Relatively weak FP mRNA expression was detected on day 5 of dioestrus. It had increased by day 25 and remained constant thereafter. In situ hybridization (ISH) localized FP solely to the cytoplasm of the luteal cells, suggesting that these cells are the only luteal targets of PGF2alpha in this species. Only negative results were obtained for the expression of PGFS in canine CL by routine qualitative RT-PCR. When Real Time (TaqMan) PCR was applied, repetitively more negative than positive results were obtained at all timepoints. Any positive measurements observed at any point were neither repeatable nor related to the stage of dioestrus. This led us to conclude that expression of PGFS is either absent or present at very low level only. These data suggest that luteal regression in non-pregnant bitches is not modulated by PGF2alpha. However, the FP seems to be constitutionally expressed, explaining the receptivity of canine CL to exogenous PGF2alpha.     

Treatment of multidrug resistant (MDR1) murine leukemia with P-glycoprotein substrates accelerates the course of the disease

The prolactin (PRL) surge in cycling rats during the proestrous afternoon is an inducer of apoptotic cell death in luteal cells. This luteolytic action of PRL is peculiar, because PRL may be categorized as a survival factor, if other known physiological functions of PRL are taken into account. Here we analyzed the underlying molecular/cellular mechanisms of this PRL-induced apoptosis. Corpora lutea (CL) were prepared from the ovary on the proestrous day and cultured with or without PRL (2 microg/ml). An addition of PRL to the culture medium induced DNA breakdown in the nuclei of cells mostly identified as steroidogenic by 3beta-HSD activity staining, and the number of 3beta-HSD-positive cells were significantly decreased, indicating the induction of apoptotic cell death by PRL among luteal cells in culture. Next, the expression of membrane form-Fas ligand (mFasL) in the luteal cell lysate was quantified, because Fas receptor is known to have an exact physiological role in luteolysis. An addition of PRL increased the expression of mFasL. Immunostaining and TUNEL assay on regressing CL revealed that both CD3-positive cells and FasL-positive cells were co-localized in the regions where apoptosis convergently occurred. Moreover, an addition of concanavalin A (ConA), a T-cell specific activator, to the culture mimicked the PRL action by inducing apoptosis in luteal cells and enhancing the expression of mFasL. These data suggest that the CD3-positive T lymphocyte in the CL is at least one of the PRL-effector cell species during the process of luteolysis in rats, and that FasL expression of these cells is upregulated by PRL.     

Prolactin involvement in the regulation of the hypothalamic-pituitary-ovarian axis during the early luteal phase of the porcine estrous cycle.

Our previous in vivo and in vitro studies revealed that prolactin (PRL) affected luteal function during the first days of the porcine estrous cycle. Since the mechanism by which the luteotrophic action of PRL might be mediated was not elucidated, the goal of the present study is to investigate the effects of short term, in vivo administration of PRL on in vitro functions of hypothalamic explants, adenohypophyseal cells and luteal cells of sows. Injections of PRL or saline (performed every 2h) started shortly after the preovulatory LH surge and lasted for 2 or 3 days. Peripheral blood plasma for determination of LH, PRL and progesterone (P(4)) was sampled at 4h intervals. Ovaries, pituitaries and the stalk median eminence (SME) dissected after slaughter were used for in vitro studies. Luteal and adenohypophysial cells as well as hypothalamic tissue were incubated/cultured with different treatments. Medium and plasma levels of GnRH, LH and P(4) were quantified by radioimmunoassays (RIAs). Corpora lutea (CL) were used for LH/human chorionic gonadotrophin (hCG) receptor analysis. In vivo and in vitro treatment with PRL increased the in vitro GnRH release by hypothalamic explants (P<0.05). GnRH-stimulated LH production was enhanced in PRL-treated sows compared to that of control sows (P<0.05). PRL injections had no effect on plasma P(4) concentrations during the treatment period. However, luteal secretion of P(4) (P=0.06) and LH/hCG receptor concentration (P=0.079) tended to be higher in PRL-treated sows in comparison to those of controls. The results indicate that PRL may be involved in the regulation of the hypothalamic-pituitary-ovarian axis at the beginning of the luteal phase of the porcine estrous cycle.     

Aglepristone (RU534) administration to non-pregnant bitches in the mid-luteal phase induces early luteal regression

The effect of the antiprogestagen aglepristone (10 mg/kg bw), administered at days 29 and 30 following the estimated day of LH surge (day 0), on corpora lutea (CL) function was examined during the diestrus phase of non-pregnant bitches. Aglepristone shortened (P < 0.01) the luteal phase and complete luteolysis (progesterone <2 ng/mL) was observed at days 40.8 ± 3.5 and 71.5 ± 4.6 (means ± SD; n = 9/group) in treated and control bitches, respectively. Peripheral estradiol-17β concentrations declined from 91.5 ± 14.3 pg/mL at day 9 to 50 pg/mL at day 18, remaining at approximately the same levels thereafter in both treated and control bitches. Intraluteal in vitro synthesis of progesterone and estradiol-17β released by CL explanted at day 38 from control bitches (511.9 ± 285.6 and 40.7 ± 17.2 pg/mg protein, respectively) did not differ from that of treated. From day 38, intraovarian hemodynamic variables (arterial blood flow, systolic peak, and end-diastolic velocities), monitored by color-coded and pulsed Doppler, decreased more steeply (P < 0.01) in aglepristone-treated (n = 4) than in control (n = 4) bitches, whereas the resistance index increased (P < 0.01) in treated animals. All the blood flow parameters were undetectable at 60 ± 3.6 and 68 ± 2.0 days (medians ± SD) after LH peak in treated and control bitches, respectively. In conclusion, aglepristone administration to dogs during the mid-luteal phase markedly accelerates the luteolytic process which is accompanied by a parallel decline in ovarian blood flow supply with a shift from approximately 8 to 10 days.     

Premature regression of the corpora lutea in superovulated cows

Morphological and functional regression of the CL was shown by $\text{15}\text{55}$ (27%) superovulated dairy cows at slaughter on D 14. Neither the routine injection of a PMSG-antiserum at the induced superovulatory oestrus, nor intrauterine infections proved to be responsible. Animals with regressed CL however had significantly lower progesterone levels in the peripheral blood at the time PMSG (D 10, P = 0,05) and prostaglandin (D 12, P = 0,02) were injected, as compared to superovulated animals with normal CL. At the induced superovulatory heat, the differences between the 2 groups disappeared. From D 4 after superovulation the differences in progesterone concentration between the 2 groups became significant again (P = 0,02 – 0,05). It is not yet clear whether the observed luteal dysfunction is due to an inadequate luteotrophic stimulus or to a premature luteolysis. The luteal regression could be caused either by a genuine luteal insufficiency of the midcyle CL or by poor heat detection.     

The expression of prolactin and its cathepsin D-mediated cleavage in the bovine corpus luteum vary with the estrous cycle

In the corpus luteum (CL), blood vessels develop, stabilize, and regress. This process depends on the ratio of pro- and antiangiogenic factors, which change during the ovarian cycle. The present study focuses on the possible roles of 23,000 (23K) prolactin (PRL) in the bovine CL and its antiangiogenic NH(2)-terminal fragments after extracellular cleavage by cathepsin D (Cath D). PRL RNA and protein were demonstrated in the CL tissue, in luteal endothelial cells, and in steroidogenic cells. Cath D was detected in CL tissue, cell extracts, and corresponding cell supernatants. In the intact CL, 23K PRL levels decreased gradually, whereas Cath D levels concomitantly increased between early and late luteal stages. In vitro, PRL cleavage occurred in the presence of acidified homogenates of CL tissue, cells, and corresponding cell supernatants. Similar fragments were obtained with purified Cath D, and their appearance was inhibited by pepstatin A. The aspartic protease specific substrate MOCAc-GKPILF~FRLK(Dnp)-D-R-NH(2) was cleaved by CL cell supernatants, providing further evidence for Cath D activity. The 16,000 PRL inhibited proliferation of luteal endothelial cells accompanied by an increase in cleaved caspase-3. In conclusion, 1) the bovine CL is able to produce PRL and to process it into antiangiogenic fragments by Cath D activity and 2) PRL cleavage might mediate angioregression during luteolysis.     

Proliferation of Luteal Steroidogenic Cells in Cattle

The rapid growth of the corpus luteum (CL) after ovulation is believed to be mainly due to an increase in the size of luteal cells (hypertrophy) rather than an increase in their number. However, the relationship between luteal growth and the proliferation of luteal steroidogenic cells (LSCs) is not fully understood. One goal of the present study was to determine whether LSCs proliferate during CL growth. A second goal was to determine whether luteinizing hormone (LH), which is known have roles in the proliferation and differentiation of follicular cells, also affects the proliferation of LSCs. Ki-67 (a cell proliferation marker) was expressed during the early, developing and mid luteal stages and some Ki-67-positive cells co-expressed HSD3B (a steroidogenic marker). DNA content in LSCs isolated from the developing CL increased much more rapidly (indicating rapid growth) than did DNA content in LSCs isolated from the mid CL. The cell cycle-progressive genes CCND2 (cyclin D2) and CCNE1 (cyclin E1) mRNA were expressed more strongly in the small luteal cells than in the large luteal cells. LH decreased the rate of increase of DNA in LSCs isolated from the mid luteal stage but not in LSCs from the developing stage. LH suppressed CCND2 expression in LSCs from the mid luteal stage but not from the developing luteal stage. Furthermore, LH receptor (LHCGR) mRNA expression was higher at the mid luteal stage than at the developing luteal stage. The overall results suggest that the growth of the bovine CL is due to not only hypertrophy of LSCs but also an increase in their number, and that the proliferative ability of luteal steroidogenic cells decreases between the developing and mid luteal stages.     

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.