Leptin Gene and Protein Expression in the Ovary During the Oestrous Cycle and Early Pregnancy in Pigs

Leptin, the product of the obese gene, is the hormone originally identified in adipocytes. It is involved in the control of satiety and energy metabolism. More recent observations suggest that leptin plays an important role in reproduction. Leptin mRNA and protein have been found in the human and the murine ovary. However, the expression of leptin in the porcine ovary has not been examined. Therefore, the aim of the present work was to compare the expression levels of porcine leptin mRNA by semiquantitative RT‐PCR and in situ hybridization, as well as leptin protein by Western blotting in the corpus luteum (CL) and ovarian stroma (OS) during mid‐ and late‐luteal phase of the oestrous cycle as well as during days 14–16 and 30–32 of pregnancy. Leptin gene and protein expression in CL was increased on days 14–16 of the cycle compared with pregnant animals. Leptin gene expression in OS was higher during the late‐luteal phase of the cycle than on days 30–32 after conception. However, comparison of leptin protein expression in OS between days 14–16 of the cycle and days 30–32 of pregnancy indicates a higher protein expression during pregnancy. Moreover, leptin gene expression was higher in porcine CL and OS on days 14–16 of pregnancy in comparison to days 30–32. Contrary to leptin mRNA expression, a higher leptin protein expression was observed on days 30–32 compared with days 14–16 after conception. In summary, the present study provides the first evidence that leptin mRNA and protein occur in porcine ovary and vary during the oestrous cycle and pregnancy. Moreover, the obtained results indicate that also locally synthesized leptin may participate in the control of pig reproduction by exercising its action at the ovarian level.     

Sialomucin Complex (Muc4) Expression in Porcine Endometrium During the Oestrous Cycle and Early Pregnancy

The non‐invasive type of implantation in the pig is characterized by the maintenance of a thick glycocalyx coating on the uterine epithelial surface microvilli. Present study investigated the alteration in the sialomucin complex (Muc4) expression during the oestrous cycle and early pregnancy in the pig. Endometrial tissue samples were immunostained with the primary antibody to the Muc4 transmembrane subunit ASGP‐2. Muc4 immunostaining increased in the surface and glandular epithelia between days 5 and 10 of oestrous cycle. Immunostaining continued to increase on day 12 with the greatest intensity of uterine Muc4 immunostaining detected on day 15 of the oestrous cycle and early pregnancy. Endometrial Muc4 expression in cyclic gilts decreased dramatically during early proestrous but continued to remain abundant in the surface and glandular epithelium of pregnant gilts during the period of conceptus attachment to the uterine surface.     

Effect of Complex Vertebral Malformation on Luteal Function in Holstein Cows During Oestrous Cycle and Early Pregnancy

The reason why cows carrying the mutation of complex vertebral malformation (CVM) show poor reproductive capability although they carry only one mutant allele is still not fully understood. Monitoring the progesterone profiles during oestrous cycle and early pregnancy in carrier cows might help explain their lowered reproductive capability. Progesterone concentration was measured in 19 CVM carrier cows and 21 control cows during oestrous cycle and early pregnancy. Milk samples were collected from all cows starting on the day of artificial insemination until day 45 post‐AI. Progesterone was measured in skim milk using enzyme‐linked immunosorbent assay (ELISA). Progesterone concentration was significantly reduced on day 7 (p < 0.05) and day 9 (p < 0.01) post‐insemination in conceived CVM carrier cows when compared with that in control conceived cows. The mean progesterone concentration during early pregnancy was significantly lower (p < 0.05) in conceived cows with CVM than that of control cows in the same period. However, the mean progesterone concentration did not differ significantly (p = 0.072) in CVM cows that showed fertilization failure or embryonic death than that of control cows. Additionally, of 13 conceived control cows, eight cows (61.5%) showed normal luteal function. In contrast, of nine conceived CVM cows, only four cows (44.4%) showed normal luteal function. The conception rate was 47.4% in CVM carrier cows and 61.9% in control cows, but this difference did not reach significance. In conclusion, progesterone concentration might be lowered during early pregnancy in conceived CVM cows compared with that in control cows.     

Expression of Aquaporin Water Channels in Equine Endometrium is Differentially Regulated During the Oestrous Cycle and Early Pregnancy

The expression of 12 different aquaporin subtypes in equine endometrium was examined at the mRNA and protein level. Endometrial samples were obtained during anoestrus, oestrus, 8, and 14 days after ovulation in non‐pregnant mares, and 14 days after ovulation in pregnant mares. Quantitative PCR revealed a time‐dependent pattern for all aquaporin subtypes examined except for AQP10 and 12. AQP3, 5 and 7 showed highest mRNA abundance 8 days after ovulation, while AQP0 and 2 were most abundant at Day 14 of the cycle in non‐pregnant mares. At 14 days of pregnancy, AQP1, 4, 8, 9 and 11 displayed highest expression levels. Western blot analysis confirmed protein expression of AQP0, 2 and 5. Immunohistochemistry localized protein expression to luminal and glandular epithelial and stromal cells. AQP0 staining intensity was highest in samples obtained on Day 14 of the oestrous cycle. AQP2 immunoreactivity seemed to be stronger in samples collected 14 days after ovulation from non‐pregnant animals, in particular luminal epithelial staining. Samples collected 8 days after ovulation from cyclic animals were characterized by intense AQP5 staining of glandular epithelium, predominantly in the deeper glands. Progesterone treatment of anoestrous mares did not enhance expression of AQPs, indicating that factors other than progesterone are required for the up‐regulation of certain AQP subtypes during dioestrus. In conclusion, it seems that an equine‐specific collaboration of aquaporin subtypes contributes to changes in endometrial fluid content occurring throughout the oestrous cycle and contributes to endometrial receptivity during early pregnancy in the mare.     

Investigations on the Endometrial Response to Intrauterine Administration of N‐Acetylcysteine in Oestrous Mares

In mares, mating‐induced persistent endometritis contributes to low fertility. The condition is in part related to delayed clearance of mucus accumulated within the uterine lumen. The objective of this study was to investigate the endometrial response of healthy mares to intrauterine (i.u.) treatment with N‐acetylcysteine (NAC). Oestrous mares (n = 12) were randomly assigned to a treatment (TM) or control (C) group and received an i.u. infusion of 5% NAC and saline (total volume 140 ml), respectively. Endometrial biopsies were collected in five of the mares 24 h after treatment, in the remaining seven mares 72 h after treatment. Endometrial biopsies were evaluated for integrity of the luminal epithelium, number of polymorphonuclear neutrophils (PMN), staining for cyclooxygenase 2 (COX2), staining with Kiel 67 antigen (Ki‐67), lectins and periodic acid‐Schiff (PAS). The integrity of endometrial epithelial cells was not affected by treatment (no statistical differences between groups or times). At 24 h after treatment, the mean number of PMN in endometrial biopsies from NAC‐ and C‐mares did not differ, but at 72 h after treatment, number of PMN was significantly higher (p < 0.05) in C (3.9 ± 0.6 PMN/field) compared with NAC‐treated mares (2.3 ± 0.2 PMN/field). At 72 h after treatment, the intensity of staining for COX2 was significantly higher after saline than after NAC treatment (p < 0.05). In the epithelium, no differences in staining for the proliferation marker Ki‐67 were seen with respect to time and treatment. Score for the lectin wheat germ agglutinin (WGA) was slightly higher in NAC‐treated mares than in C‐mares 72 h after treatment (p < 0.05). Score for PAS staining of mucus in deep uterine glands differed significantly between groups at 24 h after treatment (p < 0.05). The present study demonstrates that NAC does not adversely affect the endometrial function. Moreover, an anti‐inflammatory effect on the equine endometrium was observed.     

Urinary Oestrogens in Heifers Treated with Prednisolone During the Oestrous Cycle

Heiferis treated with a glucocorticoid during the oestrous cycle, displayed increased follicular activity and small corpora lutea compared to untreated controls (Tomasgard 1976a), but showed no signs of oestrogen production during the treatment.     

Histological Study of Canine Mammary Gland During the Oestrous Cycle

The development of the mammary gland is a puzzling phenomenon and the research on this field has been focussed mostly on the carcinogenesis, with a less goal‐oriented concern in basic histology. In order to determine the histological features of normal mammary gland in the different oestrous phases we used 39 non‐pregnant female dogs of various breeds and ages. The animals were grouped in: pre‐pubertal, pro‐oestrous, oestrous, early and late dioestrous, early and late anoestrous phases. Major changes of the canine mammary histology throughout the oestrous cycle were identified in this study. A rudimentary gland with few ducts in the base of the nipple was observed in pre‐pubertal female individuals and pubertal pro‐oestrous female ones. In the oestrus, small inactive lobules associated with ductal branching and inconspicuous regressive changes were observed, while in early dioestrus, a ductal arborization was present. In late dioestrus, a complete lobuloalveolar differentiation and secretory capacity was achieved. The regressive histological features were abundant on early anoestrus, and markedly generalized on late anoestrus. The regressive process was longer in the more caudal gland pairs. This work provides baseline knowledge of canine mammary gland that may be relevant for interspecies comparative purposes and for pathologists dealing with mammary gland tumours.     

Plasma Progesterone in Gilts During the Prepuberal Period and the First Oestrous Cycle

The progesterone concentration of peripheral plasma in pigs has previously been studied during the oestrous cycle and in early pregnancy (Tillson & Erb 1967, Stabenfeldt et al 1969). The present study was undertaken to obtain some information on the concentration of plasma progesterone during the prepuberal period and during the first oestrous cycle in the pig.     

Accumulation of Fluid in the Infundibulum During the Estrous Cycle in Mares

The funnel-shaped cranial portion (infundibulum) of the oviduct is contiguous with the ovulation fossa in mares. An accumulation of fluid in the infundibular area was discovered by transrectal ultrasonic imaging and was studied daily in both oviducts of 12 mares from day –10 to day 10 (day 0 = ovulation), and from day –6 to day 6 during 35 estrous cycles of young, intermediate, and old mares (n = 8 mares/group). The infundibulum was identified by processes (fimbriae) and folds in the pocket of fluid. The amount of fluid accumulation was scored from 0 to 3 (nil to maximum). Frequency of detection of fluid in the infundibular area increased between day –10 (46% of oviducts) and day –3 (88%), and decreased between day –3 and day 7 (8%; P < .002). The day-to-day profile for changes in the score for amount of fluid was significant (P < .0001) and similar to the profile for frequency of detection of the infundibulum. The profiles for the two infundibular end points and scores for endometrial echotexture (an indicator of edema) were similar to the reported profile for systemic estradiol concentrations. The frequency of infundibulum detection was greater (P < .0009) for the side ipsilateral to the preovulatory follicle and ovulation (51%) than for the opposite side (36%). No difference among ages was found for either oviductal end point. Results indicated that changes in the amount of fluid accumulation in the infundibular area and endometrial edema were estrous cycle dependent and similar to previously reported changes in systemic concentrations of estradiol.     

Expression of Adiponectin and its Receptors in the Porcine Hypothalamus During the Oestrous Cycle

Adiponectin is a hormonal link between obesity and reproduction, and its actions are mediated by two types of receptors: adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2). This study compares the expression levels of adiponectin and adiponectin receptor mRNAs and proteins in selected areas of the porcine hypothalamus responsible for GnRH production and secretion: the mediobasal hypothalamus (MBH), pre‐optic area (POA) and stalk median eminence (SME). The tissue samples were harvested on days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle. Adiponectin mRNA expression in MBH was significantly lower on days 14–16, whereas in SME, the most pronounced gene expression was found on days 2–3 of the cycle (p < 0.05). Adiponectin protein in MBH was most abundant on days 17–19 and in POA on days 2–3 (p < 0.05). Adiponectin protein expression in SME was at similar level throughout the most of the cycle with a statistically significant drop (p < 0.05) on days 14–16. AdipoR1 gene expression in POA was potentiated on days 2–3 and 10–12 of the oestrous cycle (p < 0.05). In SME, the highest AdipoR1 mRNA expression was noted on days 2–3 (p < 0.05). The concentrations of the AdipoR1 protein in POA were similar throughout the luteal phase (days 2–14 of the cycle), and they decreased on days 17–19 (p < 0.05). In SME, AdipoR1 protein expression peak occurred on days 2–3 (p < 0.05). The expression patterns of the AdipoR2 gene in MBH, POA and SME revealed the highest mRNA levels on days 2–3 of the cycle (p < 0.05). The highest content of AdipoR2 protein in MBH was reported on days 2–3 (p < 0.05), while in POA on days 17–19 and in SME on days 10–12 and 14–16 (p < 0.05). This study demonstrated that adiponectin and adiponectin receptor mRNAs and proteins are present in the porcine hypothalamus and that their expression levels are determined by the pig's endocrine status related to the oestrous cycle.     

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 (P₄) and oestradiol-17β. 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 P₄ > 1 ng/ml. CHs were also observed in the group, which had low level of P₄ (P₄ < 1 ng/ml). CAs were found regardless of the P₄ 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.     

Evaluation of von Willebrand Factor During Pregnancy,Lactation and Oestrous Cycle in Bitches Affected and Unaffected by von Willebrand Disease

Plasmatic concentrations of von Willebrand Factor (vWF) increase during pregnancy in humans and dogs; however the mechanism of such increase is still not well defined. The aims of this study were: (i) to evaluate changes in vWF concentration during pregnancy and during the subsequent oestrous cycle in bitches affected and unaffected by von Willebrand Disease (vWD); (ii) to correlate the vWF levels and cortisol levels in both groups. Seven vWD affected (GI) and nine unaffected (GII) bitches were used. The animals were assessed during pregnancy, parturition, lactation and non‐gestational oestrous cycle in 11 moments (Pregnancy 1, Pregnancy 2, Parturition, Lactation 1, Lactation 2, Lactation 3, Anestrus, Proestrus, Oestrus, Diestrus 1, and Diestrus 2). The following tests were performed; measurement of von Willebrand factor antigen (vWF:Ag), albumin and cortisol. In both groups, vWF concentration remained stable during the non‐gestational oestrous cycle, but increased during pregnancy, with the highest value observed at parturition. Increases of 70% and 124% in vWF were seen in GI and GII, respectively, compared to anestrus. No correlation was found between vWF and cortisol. Values of vWF:Ag changed during pregnancy, with a peak at parturition, both in vWD affected and unaffected animals. Values of vWF were not altered in the different phases of the oestrous cycle following pregnancy in both groups. Evaluation of vWF during pregnancy can cause false negative results for vWD, but assessment can be performed at any point in the oestrous cycle of non‐pregnant bitches.     

In vitro Release of Ovarian Progesterone is Decreased During the Oestrous Cycle and Pregnancy of Swine with Obesity/Leptin Resistance

Previous studies indicate that reproductive prolificacy of obese swine breeds is markedly influenced by embryo losses in early pregnancy. In such period, adequate secretion of progesterone (P4) by the ovary is essential for pregnancy success. This study analyses the luteal functionality during the oestrous cycle and early pregnancy of Iberian sows and Large White x Landrace females, in terms of P4 secretion after in vitro culture of luteal tissue stimulated or not with luteinizing hormone (LH). The secretion of progesterone (expressed in ng/mg of luteal tissue or ng/mgLT) of the corpora lutea of obese Iberian swine was always hampered when compared to lean genotypes, either during early oestrous cycle (110.7 ± 37.8 vs 259.7 ± 10.2 ng/mgLT; p < 0.0001), late oestrous cycle (49.0 ± 3.5 vs 75.92 ± 7.14 ng/mgLT; p < 0.0001) or early pregnancy (38.4 ± 2.1 vs 70.7 ± 5.3 ng/mgLT; p < 0.0001). The differences in basal P4 secretion remained after stimulation with LH. Finally, P4 secretion during early pregnancy of Iberian sows decreased with age and, hence, with obesity features (46.6 ± 4.2 vs 65.5 ± 4.8 ng/mgLT; p < 0.001). In conclusion, the results of the present study provide convincing evidence of a reduced luteal function during oestrous cycle and early pregnancy of sows with obesity/leptin resistance like Iberian sows, which may contribute to the low reproductive efficiency reported in this breed.     

Size Distribution of Dispersed Luteal Cells During Oestrous Cycle in Angora Goats

The present study examines the size distribution of the goat steroidogenic luteal cells throughout the oestrous cycle. Corpora lutea (CL) were collected after laparatomy on days 5, 10 and 16 of the oestrous cycle. Luteal cells were isolated from CL by collagenase digestion. Steriodogenic luteal cells were identified by staining of the cells for 3beta-hydroxysteroid dehydrogenase activity, a marker for steroidogenic cells. Luteal cells having steroidogenic capacity covered a wide spectrum of sizes, ranging from 5 to 37.5 microm in diameter. There was a significant increase in mean cell diameters (p < 0.01) as CL aged. The mean cell diameter on day 5 was 11.55 +/- 0.12 microm, which was significantly increased and reached up to 19.18 +/- 0.24 mum by day 16 of the oestrous cycle. The ratio of large to small luteal cells was 0.06:1.0 on day 5 of the oestrous cycle. This ratio increased to 0.78:1.0 by day 16 of the oestrous cycle. Luteal cell size on days 5, 10 and 16 of the oestrous cycle reached its maximum at 7.5, 10 and 35 microm in diameter, respectively. Development of CL is associated with an increase in luteal cell size in goats. It is likely that small luteal cells could develop into large luteal cells as CL becomes older during oestrous cycle in goats.     

hCG‐Induced Ovulation in Thoroughbred Mares Does Not Affect Corpus luteum Development and Function During Early Pregnancy

Our aim was to compare Corpus luteum (CL) development and blood plasma concentration of progesterone ([P₄]) in thoroughbred mares after spontaneous (Control: C) or human chorionic gonadotrophin (hCG)‐induced ovulation. Lactating mares (C = 12; hCG = 21) were daily teased and mated during second oestrus post‐partum. Treated mares received 2500 IU hCG i.v. at first day of behavioural oestrus when dominant follicular size was >35, ≤42 mm and mated 12–24 h after. Control mares in oestrus were mated with dominant follicular size ≥45 mm. Dominant follicle before ovulation, CL and gestational sac were measured by ultrasound and [P₄] by radioimmunoassay (RIA). Blood sampling and ultrasound CL exams were done at days 1, 2, 3, 4, 8, 12, 16, 20, 25, 30, 35, 40, 45, 60 and 90 after ovulation and gestational sac from day 12 after ovulation in pregnant (P) mares; non‐pregnant (NP) were followed until oestrus returned. Data analyses considered four subgroups: hCG‐P, hCG‐NP, C‐P and C‐NP. Preovulatory follicular size was smaller in hCG mares than in C: 39.2 ± 2.7 mm vs 51.0 ± 1.8 mm (p < 0.0001). All hCG mares ovulated 24–48 h after treatment and presented similar oestrus duration as controls. C. luteum size in P mares showed the same pattern of development through days 4–35, presenting erratic differences during initial establishment. Thus, on days 1 and 3, CL was smaller in hCG‐P (p < 0.05); while in hCG‐NP, CL size was greater than in C‐NP on day three (p = 0.03). Corpus luteum size remained stable until day 90 in hCG‐P mares, while in C‐P a transient and apparently not functional increase was detected on days 40 and 45 (p < 0.05) and the decrease from day 60 onwards, made this difference to disappear. No differences were observed in [P₄] pattern between P, or between NP subgroups, respectively. So, hCG‐induced ovulation does not affect CL development, neither [P₄] during early pregnancy. One cycle pregnancy rate tended to be lower in hCG mares while season pregnancy rates were similar to controls.     

Reproduction in the European Mink, Mustela lutreola: Oestrous Cyclicity and Early Pregnancy

Despite efforts undertaken to conserve the endangered European mink, its reproduction is still poorly studied. The aim was to study its reproductive cyclicity, faecal progesterone concentration and ovarian changes during early pregnancy, with the emphasis on the pre-implantation period and implantation. During the 2004 breeding season, oestrous cycle was monitored in 39 females as well as ovarian changes during early pregnancy in 22 females. During the 2007 breeding season, faecal progesterone concentration measured by radioimmunoassay was monitored during pregnancy in 10 females throughout their pregnancy. The breeding season 2004 started on March 18 and ended on May 10, with the peak recorded in April. The duration of first oestrus was 1–12 days. If not mated, the vast majority of females entered second oestrus after 12–55 days. In general, relatively low faecal progesterone values were detected in European mink; an average of 42.69 ± 4.70 ng/g faeces in oestrous females with a maximum of 176.44 ± 23.01 ng/g faeces on pregnancy day 12. anova indicated a significant effect of the pregnancy stage. Post hoc comparisons with Fisher least significant difference (LSD) test revealed that faecal progesterone concentrations on days 8 and 12 post coitum (p.c.), but not at the end of pregnancy (day 40), were higher when compared with the initial oestrous level. Implantation in this species occurs on day 12 p.c. and was indicated by prominent uterine swellings and failure to flush the uterine horns beyond this day. Advanced luteogenesis was observed with prominent corpora lutea found in ovaries around the time of implantation. To conclude, European mink is a seasonally polyoestrous species; the early pregnancy of European mink resembles that of European polecat, i.e. in both species, implantation occurs on day 12 p.c. without any implantation delay.     

Ovarian Steroid Regulation of Endometrial Phospholipase A2 Isoforms in Horses

Real‐time PCR was used to investigate the role of progesterone (P4) and oestradiol (E2) in regulation of endometrial cytosolic, secretory and calcium‐independent phospholipase A2 (PLA2G4A, PLA2G2A and PLA2G6, respectively) gene expression. Ovariectomized mares underwent 6 days of E2 pre‐treatment followed by 14 days of P4 supplementation. At the start of P4 treatment (Day 1), mares were assigned in a 2 × 2 factorial design to receive either E2 or vehicle starting on Day 11 and endometrial biopsy collection on either Day 14 when P4 concentrations remained high (>4 ng/ml) or Day 16 when P4 concentrations had declined (0.5–2 ng/ml). Additional biopsies were collected from ovariectomized mares on Day 8, which served as control. Blood samples were collected for P4 determination. PLA2G4A expression was higher (p < 0.05) on Day 14 compared with Day 8. In contrast, PLA2G2A did not change significantly (p < 0.12). PLA2G4A and PLA2G2A gene expression increased (p < 0.05), as P4 concentration dropped, on Day 16. In contrast, PLA2G6 gene expression did not show differences between days. Treatment with oestradiol did not increase PLA2 isoforms expression when compared to treatment with the vehicle. PLA2G4A and PLA2G2A were positively correlated with each other and negatively correlated with P4 concentrations. In conclusion, P4 withdrawal upregulated PLA2G4A and PLA2G2A gene expression, and this was not affected by E2. PLA2G4A and PLA2G2A but not PLA2G6 gene expression may be involved in controlling prostaglandin F2 alpha synthesis and luteolysis.