The oviduct and uterus provide the environment for the establishment of pregnancy. Among others, growth factor systems are involved in functional signaling interactions at the pre- and peri-implantation maternal-conceptus interface in pigs. Distinct regulation of epidermal growth factor Receptor (EGF-R), vascular endothelial growth factor receptor (VEGF-R) and fibroblast growth factor receptor (FGF-R) systems and of bioactivation of EGF-R in porcine oviduct and endometrium during the estrous cycle, early pregnancy and during steroid replacement in ovariectomized gilts is summarized. Remarkable influences of ovarian steroids and EGF on the expression of specific markers of transcription and translation in these tissues are discussed. Known biological effects of the EGF, VEGF and FGF are related to cellular differentiation and angiogenesis. This suggests their involvement in the transformation of the endometrium into a decidua subsequently leading towards successful establishment of pregnancy. Peripheral steroids may exert their effects on epithelial cells both in a direct genomic manner or through mediators such as growth factors. The aim of our study was to draw specific attention to the paracrine regulation in the porcine endometrium especially during the implantation window.

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Previous studies have suggested the presence of a glucose-sensing mechanism in the hindbrain that appears to regulate reproductive function as well as feeding behavior. The ependymocytes lining the ventricular wall of the hindbrain showed immunoreactivities to pancreatic glucokinase (GK), a key enzyme for glucose sensing in pancreatic B cells. Our goal in the present study was to test whether the GK-immunopositive ependymocytes in the wall of the fourth cerebroventricle (4V) play a role in regulating gonadal activity. Our approach was to determine the effect of injecting alloxan, a GK inhibitor, into the 4V on pulsatile luteinizing hormone (LH) secretion. Estrogen-primed ovariectomized rats received an injection of alloxan (10 or 20 μg/animal) into the 4V and blood samples were collected every 6 min for 3 h for measurement of blood LH, corticosterone and glucose levels. Pulsatile LH secretion was suppressed after alloxan injection and all pulse parameters were significantly (P<0.05) inhibited by 20 μg alloxan. Plasma corticosterone levels were increased significantly (P<0.05) by 20 μg alloxan, suggesting that LH pulse suppression by alloxan may be at least partly mediated by activation of the hypothalamo-pituitary-adrenal axis. The present results suggest that acute suppression of GK activity in the hindbrain inhibits pulsatile LH secretion in female rats, and supports the idea that GK-immunopositive ependymocytes may sense glucose levels in the cerebrospinal fluid and play a role in regulation of LH secretion.

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The effects of delipidation and the oxygen (O₂) concentration in the atmosphere during culture on in vitro development and H₂O₂ content were investigated in porcine in vivo fertilized embryos and embryos after in vitro maturation and in vitro fertilization (IVM/IVF embryos). There was no significant difference in the developmental rates to the blastocyst stage between the intact and delipidated IVM/IVF embryos. However, the mean number of cells in blastocysts derived from delipidated IVM/IVF embryos (19.8 ± 0.8 cells) was significantly smaller than that from intact embryos (24.2 ± 1.2 cells). Although there were no significant differences in the developmental rates to the blastocyst stage of intact and delipidated IVM/IVF embryos between the cultures under 5% O₂ and 20% O₂, the developmental rate of intact IVM/IVF embryos cultured under 5% O₂ (27.1%) was significantly higher than that of the delipidated embryos cultured under 20% O₂ (19.3%). On the other hand, there was no difference in the developmental rate to the blastocyst stage between in vivo fertilized embryos cultured under 5% O₂ and 20% O₂. Hydrogen peroxide (H₂O₂), one of the reactive oxygen species (ROS), is thought to cause damage to embryos. The H₂O₂ content per embryo derived from oocytes cultured under 5% O₂ (in vivo fertilized, 58.0 ± 2.5 pixels; IVM/IVF, 79.6 ± 3.2 pixels) was significantly lower than that (in vivo fertilized, 100.2 ± 3.8 pixels; IVM/IVF, 103.9 ± 3.2 pixels) under 20% O₂. Furthermore, the level of H₂O₂ in delipidated IVM/IVF embryos (94.7 ± 3.9 pixels) was significantly lower than that in intact embryos (103.9 ± 3.2 pixels) cultured under 20% O₂. The present results indicate that the delipidation of porcine IVM/IVF embryos and reduction of the O₂ concentration decreased the H₂O₂ level rather than the in vitro developmental rate to the blastocyst stage.

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Equine chorionic gonadotropin (eCG), which consists of highly glycosylated α- and β-subunits, is a unique member of the gonadotropin family because it elicits response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in species other than the horse. In this study, recombinant tethered-eCG as well as its deglycosylated mutants were produced to determine if α- and β- subunits can be synthesized as a single polypeptide chain (tethered-eCG) and display biological activity. We found that tethered-eCG (T- βα) had both LH- and FSH-like activities comparable to dimeric eCG. Luteinizing hormone-like activity of tethered-eCGs deglycosylated at Asn⁵⁶ (T-βα56) was decreased. In contrast, LH-like activity of eCG without O-glycosylated carboxyl-terminal peptide (CTP) (T-βcα) was slightly decreased but still similar to T-βα. Double mutation at Asn⁵⁶ and CTP (T-βcα56) caused marked decrease in the activity, indicating that both glycosylations at Asn⁵⁶ and CTP are involved in LH-like activity in the tethered form. Interestingly, FSH-like activity remained in all deglycosylated eCG mutants (T-βα56, T-βcα and T-βcα56) as well as T-βα. The biological roles of oligosaccharides at Asn⁵⁶ of eCG α-subunit and O-linked peptide of β-subunit appear to be different in LH- and FSH-like activities in tethered-eCG.

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The present study examined the effects of 17 β-estradiol (E₂) on in vitro maturation and subsequent in vitro fertilization of pig oocytes matured with or without cumulus cells. When E₂ (10 ng/ml) was added to the protein-free maturation medium, the proportions of cumulus-enclosed oocytes that underwent germinal vesicle breakdown and reached metaphase II were significantly reduced (P<0.05), and cumulus expansion was also significantly inhibited (P<0.05) compared with the control (no E₂ added). Although oocytes matured in the presence of E₂ were penetrated by sperm in vitro at the same level as the control, the incidences of male pronuclear (MPN) formation and activated oocytes were significantly lower (P<0.05) than the control. These inhibitory effects of E₂ were prevented when the medium was supplemented with E₂ together with its antagonist, ICI 182,780 (1 μg/ml), although the presence of the antagonist alone in the medium had no effect on the maturation and fertilization in vitro of oocytes. In cumulus-free oocytes, E₂ had no effect on nuclear maturation and penetration in vitro, but low MPN formation was observed in oocytes matured in the presence and absence of E₂. When cumulus-enclosed oocytes were cultured in the presence of progesterone (P₄; 600 ng/ml) alone or together with E₂, no significant differences in nuclear maturation, cumulus expansion or penetration in vitro were observed compared with control oocytes. The concentration of P₄ in maturation medium was significantly (P<0.01) lower when cumulus-enclosed oocytes were cultured for 44 h in the medium with E₂ than in medium without E₂. These results indicate that E₂ inhibits both nuclear and cytoplasmic maturation of cumulus-enclosed pig oocytes, and that this inhibition can be prevented by an E₂ antagonist or P₄. This E₂ inhibition may occur indirectly via the cumulus cells and inhibition of P₄ synthesis.

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This study aimed to describe the changes in the thecal vasculature during ovarian follicular atresia in the swamp buffalo. Ovaries of Philippine swamp buffalo (Bubalus bubalis; SB), crossbred (SB × Murrah buffalo; CB) and Holstein-Friesian cow (Bos taurus; HF) were collected from slaughterhouses, fixed in 10% formalin in PBS and embedded in paraffin. Sections of healthy follicle and various follicular stages of atresia were stained with Bandeiraea simplicifolia-I lectin (BSL-I) to visualize the endothelial cells of blood vessels. In the theca interna, healthy follicles in SB had a significantly lower number of capillary vessels than other breeds and other atretic stages of follicle. From healthy to early atretic follicle, theca interna in all breeds showed a significant decrease in the area of capillary vessel. Capillary vessel area significantly increased (but was smaller than in healthy follicle) in the middle stage of atresia and declined again in the late atretic follicle (greater than in early atresia but smaller than in healthy follicle) in SB only. No significant change in the capillary vessel area of theca interna was noted in both CB and HF from early to late atretic follicles. There was no significant difference in the capillary vessel number and area of theca externa among the different breeds and atretic stages of follicle. These results suggest that there are dynamic changes occurring in the thecal vasculature of SB but not CB during follicular atresia which differs among cattle.

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20α-Hydroxysteroid dehydrogenase (20α-HSD), which catalyzes the conversion of progesterone to its inactive form 20α-dihydroprogesterone, is expressed in murine placenta and has been suggested to play roles in maintaining pregnancy. To understand the role of 20α-HSD during pregnancy in the goat, as a first step, cloning and sequencing of 20α-HSD cDNA were performed. The full nucleotide sequence of 20α-HSD cDNA was determined on samples obtained from the corpus luteum at the luteal phase of the estrous cycle and the placenta in late pregnancy by RT-PCR and 3' and 5' RACE systems. Cloned 20α-HSD cDNA consisted of 1124 bp and belonged to the aldo-keto reductase superfamily. From the start codon to stop codon there were 323 amino acids, the same as in other species. To verify whether the protein derived from goat 20α-HSD cDNA had 20α-HSD activity, the cDNA was expressed by bacteria. Bacterially expressed goat 20α-HSD protein showed 20α-HSD enzyme activity. A tissue distribution study demonstrated that 20α-HSD was expressed in the placenta, but not in the adrenal gland, liver and spleen during pregnancy. The present study suggests that goat 20α-HSD is another member of the aldo-keto reductase superfamily and that it plays a role in the placenta during pregnancy.

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The purpose of this study was to determine whether nitric oxide (NO) mediates tumor necrosis factor (TNF)α influence on the bovine endometrium. TNFα influence on the bovine endometrium is limited to the stromal cells. Therefore, it was interesting to find out whether NO production by the stromal cells, stimulated by TNFα might influence the endometrial epithelium. Moreover, we investigated the intracellular mechanisms of TNFα- and NO-regulated prostaglandin (PG) F_2α and PGE₂ synthesis. Epithelial and stromal cells from the bovine endometrium (Days 2-5 of the oestrous cycle) were separated by means of enzymatic dispersion and cultured for 6-7 days in 48-well plates. The confluent endometrial cells were exposed to a NO donor (S-NAP; 1-1000 μM) for 24 h. S-NAP strongly stimulated PGE₂ production in both bovine endometrial cell types (P<0.001). The effect of SNAP on PGF_2α production was limited only to the stromal cells (P<0.05). To study the intracellular mechanisms of TNFα and NO action, stromal cells were incubated for 24 h with TNFα or S-NAP and with NO synthase (NOS) inhibitor (L-NAME; 10 μM) or an inhibitor of phosphodiesterase (IBMX; 10 μM). When the cells were exposed to TNFα in combination with NOS inhibitor (L-NAME), TNFα-stimulated PGs production was reduced (P<0.05). The inhibition of enzymatic degradation of cGMP by IBMX augmented the actions of S-NAP and TNFα on PGs production (P<0.05). The overall results suggest that TNFα augments PGs production by bovine endometrial stromal cells partially via induction of NOS with subsequent stimulation of NO-cGMP formation. NO also stimulates PGE₂ production in epithelial cells.

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The objective of this study was to evaluate the effects of a progesterone-releasing intravaginal device (PRID) containing an estradiol benzoate capsule on ovarian dysfunction, including ovarian quiescence, follicular cyst (FC) and luteal cyst or cystic corpus luteum (LC/CCL), in postpartum dairy cows. These ovarian dysfunctions were examined by palpation per rectum relative to plasma progesterone status. The results of clinical examination and hormone assay determined ovarian quiescence in 13 cows, FC in 15 cows and LC/CCL in 7 cows. These cows were treated with PRID for 12 d and then clinical examination was performed. After PRID removal, the proportion of cows exhibiting estrous signs within 7 d and confirmed formation of CL within 7-14 d (markedly effective) were 69.2 % (n=9) for ovarian quiescence, 46.7 % (n=7) for FC, and 28.6 % (2 cows) for LC/CCL. Two cows (15.4 %) in ovarian quiescence, 5 cows (33.3%) with FC and 4 cows (57.1 %) with LC/CCL did not exhibit estrous signs but were recognized as having formed CL within 12-16 d after removal of PRID (effective). These results suggest that treatments of PRID with estradiol benzoate for 12 d have therapeutic efficacy on ovarian dysfunction including ovarian quiescence, FC and LC/CCL in postpartum dairy cows.

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Effects of maternal exposure to low doses of bisphenol A (BPA), including those comparable with human exposure levels, on growth and development of the female reproductive system and uterine carcinogenesis in Donryu rats were investigated. Dams were administered BPA (0, 0.006 and 6 mg/kg/day) daily by gavage from gestation day 2 up to the day before weaning (postnatal day 21 at offspring). The serum levels of BPA were significantly elevated in the dams receiving 6 mg/kg/day, however, BPA levels in the milk of dams, and those in the serum and liver of offspring were similar between control and treated groups. The treatment did not exert any influences on uterine development including weight, gland genesis and estrogen receptor α expression, vaginal opening and gonadotropin secretion in the female offspring up to puberty. After maturation, no effects were evident with regard to estrous cyclicity in female offspring treated with BPA. In addition, the treatment had no effects on age-related morphological changes of the reproductive and endocrine organs and uterine carcinogenesis until 15 months of age. The results demonstrate that maternal exposure to BPA at levels comparable to human exposure did not have any effects on the female reproductive system of offspring in rats. In addition, BPA was also found in the serum, milk and liver of control dams and pups, and low levels of BPA were detected in drinking water and pellet diet. The present study showed that the experimental animals were also exposed to environmental BPA in the animal room.

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To investigate how uterine inflammation affects ovarian activity in rats, endometritis was induced and changes in the length of estrous cycle and serum concentrations of estradiol-17β (E₂) and progesterone (P₄) were examined. A suspension of Staphylococcus aureus (bacterial solution) or iodine solution was infused into the uterine lumen at various estrous phases. When the bacterial solution was infused at estrus, metestrus, or the first day of diestrus, the following diestrus continued for 5 to 12 days. In the case of the iodine solution, regardless of the estrous phase of the infusion, the following diestrus continued for approximately 6 days. E₂ concentration after infusion of each solution did not fluctuate largely and remained at a low concentration (around 5 pg/ml). P₄ concentration was high (35-45 ng/ml) on the day following infusion, but decreased rapidly to base line values within a few days and remained thereafter at a low level (around 5 ng/ml). It is assumed that the endometritis caused by biological or chemical stimulation raises the concentration of P₄ to depress gonadotrophic hormone secretion, and hence this high P₄ concentration might inhibit the growth of ovarian follicles.

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We investigated the efficacy of a controlled internal drug release (CIDR) device in the reproductive management of an embryo donor beef herd. Superovulation of embryo donors was routinely induced by a combination of FSH and prostaglandin F₂α analogue (PGF₂α) at intervals of a few months, and after embryo recovery estrus of the donors was synchronized with PGF2₂α. Between 1996 and 1998, approximately 20% of donors were diagnosed as having follicular cysts every year. Twenty-eight cases of follicular cysts recorded between 1997 and 1998 were treated with a CIDR device for 14 days to evaluate the efficacy of CIDR in resolving follicular cysts in donor herds. Initial recovery was defined as the occurrence of ovulation with estrous behavior and subsequent formation of a corpus luteum after removal of the CIDR. Initial recovery was recognized in all cases (n=28). Follicular cysts did not recur after repeated embryo recovery in 64% of the initially recovered donors, but in 36% of affected donors cysts recurred after the next embryo recovery. Subsequently, with a CIDR device instead of PGF₂α, we synchronized estrus after embryo recovery in the same herd from 1999 to 2000, to investigate the ability of CIDR to prevent the initiation of follicular cysts. Of the donors used between 1999 and 2000, approximately 30% had a history of follicular cysts. Use of CIDR for estrous synchronization after embryo recovery lowered the incidence of follicular cysts to 3% in 1999 and 0% in 2000. Treatment with CIDR proved effective at resolving follicular cysts in the embryo donor beef herd and enabled re-use of donors affected with follicular cysts. CIDR is also likely to be efficacious in lowering the occurrence of follicular cysts in donor herds when it is used for estrous synchronization after embryo recovery.

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To understand the role of IGF-I in murine pregnancy, we studied the reproductive performance of IGF-I overexpressed mice. Fetal loss occurred only in the transfected uterine horn during Day 10-15 of pregnancy. The placenta appeared healthy until Day 10 of pregnancy. From Day 12, the decidua basalis of the transfected horn increased in thickness. The vascular lumen was expanded, and most of embryos were dead. Uterine Natural Killer cells did not undergo apoptosis from Day 10 to Day 15 when they usually go through apoptosis. Thus, it is likely that IGF-I plays a role in the decidual formation through regulation of uNK cells. This is the first report to demonstrate that IGF-I overexpression can cause fetal loss during murine placentation.

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