Cross-protection and cross-neutralization capacity of ancestral and VOC-matched SARS-CoV-2 adenoviral vector-based vaccines.

COVID-19 vaccines were originally designed based on the ancestral Spike protein, but immune escape of emergent Variants of Concern (VOC) jeopardized their efficacy, warranting variant-proof vaccines. Here, we used preclinical rodent models to establish the cross-protective and cross-neutralizing capacity of adenoviral-vectored vaccines expressing VOC-matched Spike. CoroVaxG.3-D.FR, matched to Delta Plus Spike, displayed the highest levels of nAb to the matched VOC and mismatched variants. Cross-protection against viral infection in aged K18-hACE2 mice showed dramatic differences among the different vaccines. While Delta-targeted vaccines fully protected mice from a challenge with Gamma, a Gamma-based vaccine offered only partial protection to Delta challenge. Administration of CorovaxG.3-D.FR in a prime/boost regimen showed that a booster was able to increase the neutralizing capacity of the sera against all variants and fully protect aged K18-hACE2 mice against Omicron BA.1, as a BA.1-targeted vaccine did. The neutralizing capacity of the sera diminished in all cases against Omicron BA.2 and BA.5. Altogether, the data demonstrate that a booster with a vaccine based on an antigenically distant variant, such as Delta or BA.1, has the potential to protect from a wider range of SARS-CoV-2 lineages, although careful surveillance of breakthrough infections will help to evaluate combination vaccines targeting antigenically divergent variants yet to emerge.

Estrogens receptors, nuclear coactivator 1 and ligand-dependent corepressor expression are altered early during induced ovarian follicular persistence in dairy cattle.

Failure of ovulation can lead to follicular persistence, one of the main components of the pathogenesis of cystic ovarian disease (COD) in dairy cattle. Follicular persistence causes the permanence of a functional follicular structure in the ovary, which alters the cyclicity of the female and causes infertility. The aim of the present study was to evaluate the expression of estrogen receptors (ESR) 1 and 2, and the coregulatory proteins NCOA1, NRIP1 and LCOR by immunohistochemistry, in antral and preovulatory/persistent follicles in a model of follicular persistence induced by low levels of progesterone, to detect incipient changes during COD development, on the expected day of ovulation (P0) and after 5 (P5), 10 (P10) and 15 (P15) days of follicular persistence. Twenty-five Holstein cows were used, which were distributed in 5 groups: control group (n = 5), group P0 (n = 5), group P5 (n = 5), group P10 (n = 5), group P15 (n = 5). ESR1 expression was lower in antral follicles of the P5 (theca), P10 and P15 (theca and granulosa) groups relative to the control group (p < 0.05), and also lower in granulosa cells of persistent follicles of the P5, P10 and P15 groups than in dominant follicles of the control group (p < 0.05), without differences in theca cells. ESR2 expression showed no differences between groups. The ESR1:ESR2 balance favored ESR2 expression along the development of persistent follicles, as from 5 days of persistence (p < 0.05). NCOA1 expression was higher in granulosa cells of both antral and persistent follicles from the P0 group relative to the P5 and P10 groups, but showed no differences with the control and P15 groups (p < 0.05). Theca cells of antral and persistent follicles showed higher expression in the P0 and P15 groups in relation to the control, P5 and P10 groups (p < 0.05). No differences were detected for NRIP1 in antral, dominant and persistent follicles between groups. LCOR expression showed a decrease in granulosa cells of antral follicles from all persistence groups relative to the control group (p < 0.05). In theca cells, antral follicles of the P10 group showed lower LCOR expression than the control group (p < 0.05). LCOR expression was similar for dominant and persistent follicles. Considering that the ESR1:ESR2 balance favored ESR2 expression along the development of persistent follicles, as well as the decreased LCOR and NCOA1 expression, we may assume that, at the early stages of persistence, there is a negative regulation of ESR transcription. This coincides with the effects of estrogens through ESR on proliferation and apoptosis among other processes that favor follicular persistence. The results obtained provide relevant information in the knowledge of local events during the development of follicular persistence that could explain the failures in the reversion of the disease through hormonal treatments and the high recurrence rates reported for COD. In addition, it contributes to the study and identification of possible therapeutic targets, for the design of new treatments.

Association between heat stress during intrauterine development and the expression and regulation of ovarian steroid hormone receptors in adult Holstein cows.

CONTEXT: Dairy cattle experience stressful environmental situations that affect production. Heat stress during gestation can influence the intrauterine development of offspring, resulting in long-term damage that can affect the reproductive life of the adult offspring. AIM: The aim of the present study was to evaluate changes in the expression and regulation of steroid hormone receptors in the ovary of Holstein cows gestated under different temperature-humidity index (THI) during their in utero development. METHODS: Animals were classified by their exposure to temperature-humidity index (THI) ≥72 during their development in utero according to date of birth or date of effective service of their mother. This study was not carried out under controlled conditions, but the conditions to which the cows were naturally exposed during their development were considered retrospectively, controlling the variables in the statistical analyses (age as a covariate, dairy farm as a random factor). Gestation was divided into two periods (P1=days 0-150; and P2=day 151 to calving) and three trimesters (T1=days 0-90; T2=days 91-180; and T3=day 181 to calving), and the exposure to THI ≥72 was calculated in each one. The following characteristics were evaluated: gene expression of estrogen receptor (ESR) 1, ESR2 and progesterone receptor (PGR), CpG methylation in the 5'UTR of ESR1 and ESR2, and protein expression of ESR1, ESR2, PGR and coregulatory proteins in the dominant follicles of daughter cows in adulthood. KEY RESULTS: We found associations between heat stress variables during gestation and the methylation status of CpG sites in the 5'UTR of ESR1 and ESR2 in dominant follicles. Results also showed association between exposure to high THI values during intrauterine development and expression of ESR1, ESR2 and PGR and coregulatory proteins in dominant follicles of adult cows. CONCLUSIONS: These results provide novel information about the impact of prenatal heat stress on molecular aspects at the ovary level in the offspring, during their adult life, which probably impacts the reproductive aspects of the herd.

Characterization of an incipient granulosa cell tumour in a Holstein cow: Steroid hormone receptors and coregulators expression.

The objective of this study was to describe a case of a granulosa cell tumour (GCT) of incipient formation and to characterize it by its immunohistochemical pattern and hormonal profile. The case presented corresponds to a 7-year-old Holstein cow without reproductive disorders. No alterations were observed at rectal palpation, neither in the ultrasonography nor in the hormonal profile. A GCT concomitant with normal follicular development was diagnosed. Through a panel of immunohistochemical markers, a highly differentiated pattern could be determined in the GCT, which preserves the expression of steroid receptors (ESR1, ESR2 and PR) typical of granulosa cells, but does not express the enzymes for the synthesis of androgens (CYP17A1) and oestrogens (CYP19A1). In addition, the expression of co-regulators of steroid hormone receptors and neuroendocrine markers was described for the first time in a GCT in cattle. These results increase the information about GCTs in cattle before the ovarian function is compromised.

Non-clinical safety assessment and in vivo biodistribution of CoviFab, an RBD-specific F(ab')2 fragment derived from equine polyclonal antibodies.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has required the urgent development of new therapies, among which passive immunotherapy is contemplated. CoviFab (INM005) is a RBD-specific F(ab')2 fragment derived from equine polyclonal antibodies. We investigate their preclinical security and biodistribution by in vivo and ex vivo NIR imaging after intravenous administration of a dose of 4 mg/kg at time 0 and 48 h. Images were taken at 1, 12, 24, 36, 48, 49, 60, 72, 84, 96, 108, 120, 132 and 144 h after the first intravenous injection. At 96 and 144 h, mice were sacrificed for haematology, serum chemistry, clinical pathology, histopathology and ex vivo imaging. The biodistribution profile was similar in all organs studied, with the highest fluorescence at 1 h after each injection, gradually decreasing after that each one and until the end of the study (144 h). The toxicology study revealed no significant changes in the haematology and serum chemistry parameters. Further, there were no changes in the gross and histological examination of organs. Nonclinical data of the current study confirm that CoviFab is safe, without observable adverse effects in mice. Furthermore, we confirm that bioimaging studies are a useful approach in preclinical trials to determine biodistribution.

Association between heat stress during intrauterine development and the calving-to-conception and calving-to-first-service intervals in Holstein cows.

Heat stress has been widely studied in relation to its effects on the production and reproduction of dairy cattle. However, the long-term effects of heat stress during intrauterine development on adult cows have been scarcely considered. Thus, the aim of this study was to evaluate possible changes in the reproductive performance of Holstein cows gestated under different values of the Temperature-Humidity Index (THI) during their intrauterine development. Data collected from a database of reproductive and productive records of 10,790 Holstein cows from the central region of Argentina and the THI data from the agrometeorological station of the Instituto Nacional de Tecnología Agropecuaria (INTA) EEA Rafaela, Santa Fe, Argentina, were used. The gestation of the cows evaluated was divided into trimesters, in which the highest THI cycles (sum of three or more consecutive days of exposure to a THI ≥72 during each trimester) and number of days with a THI ≥ 72 were calculated. The calving-to-conception and calving-to-first-service intervals of the cows evaluated were considered as reproductive variables associated with their first lactation. Generalized Linear Mixed Models were used, considering the cow's dairy farm as a random factor within the model. The exposure of the animals to environments with a THI ≥72 during the first trimester of gestation had a negative impact on the reproductive efficiency parameters analyzed. The results obtained indicate that the exposure of pregnant females to high THI values has a long-term impact on their daughters, which may contribute to a decrease in their reproductive performance, possibly through inherited epigenetic characteristics that remain in later generations through fetal programming.

New inhibitor targeting Acyl-CoA synthetase 4 reduces breast and prostate tumor growth, therapeutic resistance and steroidogenesis.

Acyl-CoA synthetase 4 (ACSL4) is an isoenzyme of the fatty acid ligase-coenzyme-A family taking part in arachidonic acid metabolism and steroidogenesis. ACSL4 is involved in the development of tumor aggressiveness in breast and prostate tumors through the regulation of various signal transduction pathways. Here, a bioinformatics analysis shows that the ACSL4 gene expression and proteomic signatures obtained using a cell model was also observed in tumor samples from breast and cancer patients. A well-validated ACSL4 inhibitor, however, has not been reported hindering the full exploration of this promising target and its therapeutic application on cancer and steroidogenesis inhibition. In this study, ACSL4 inhibitor PRGL493 was identified using a homology model for ACSL4 and docking based virtual screening. PRGL493 was then chemically characterized through nuclear magnetic resonance and mass spectroscopy. The inhibitory activity was demonstrated through the inhibition of arachidonic acid transformation into arachidonoyl-CoA using the recombinant enzyme and cellular models. The compound blocked cell proliferation and tumor growth in both breast and prostate cellular and animal models and sensitized tumor cells to chemotherapeutic and hormonal treatment. Moreover, PGRL493 inhibited de novo steroid synthesis in testis and adrenal cells, in a mouse model and in prostate tumor cells. This work provides proof of concept for the potential application of PGRL493 in clinical practice. Also, these findings may prove key to therapies aiming at the control of tumor growth and drug resistance in tumors which express ACSL4 and depend on steroid synthesis.

Developmental Programming: Prenatal Testosterone Excess on Ovarian SF1/DAX1/FOXO3.

Prenatal testosterone (T) excess, partly via androgenic programming, enhances follicular recruitment/persistence in sheep as in women with polycystic ovarian syndrome (PCOS). Decreased anti-Mullerian hormone (AMH) in early growing and increased AMH in antral follicles may underlie enhanced recruitment and persistence, respectively. Changes in AMH may be mediated by steroidogenic factor 1 (SF1), an enhancer of AMH, and dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX1), that antagonizes SF1. Another mediator could be forkhead box 03 (FOXO3) which regulates follicular recruitment/atresia. To test if androgen-programmed changes in SF1, DAX1, and FOXO3 proteins contribute to follicular defects in prenatal T-treated sheep, ovaries from control, prenatal T-, and dihydrotestosterone (DHT)-treated (days 30-90 of gestation) animals at fetal day (FD) 90, FD140, and 1 and 2 years-of-age were studied. Prenatal T increased DAX1 in granulosa cells of primordial through large preantral and theca cells of large preantral follicles at FD140 and increased SF1 in the granulosa cells of preantral and antral and theca cells of large preantral follicle at 2 years-of-age. Prenatal T increased FOXO3 only in theca cells of preantral (FD140) and antral (2 years-of-age) follicles. Prenatal DHT increased DAX1 in granulosa cells from small preantral follicles at FD140 while increasing SF1 in granulosa cells from antral follicles at 1 year-of-age. These age-dependent changes in DAX1/SF1 partly via androgen-programming are consistent with changes in AMH and may contribute to the enhanced follicular recruitment/persistence, and multifollicular phenotype of prenatal T-treated females and may be of translational relevance to PCOS.

Follicular structures of cows with cystic ovarian disease present altered expression of cytokines.

Ovulation is considered an inflammatory, cytokine-mediated event. Cytokines, which are recognized as growth factors with immunoregulatory properties, are involved in many cellular processes at the ovarian level. In this sense, cytokines affect fertility and are involved in the development of different ovarian disorders such as bovine cystic ovarian disease (COD). Because it has been previously demonstrated that ovarian cells represent both sources and targets of cytokines, the aim of this study was to examine the expression of several cytokines, including IL-1ß, IL-1RA, IL-1RI, IL-1RII, IL-4 and IL-8, in ovarian follicular structures from cows with spontaneous COD. The protein expression of these cytokines was evaluated by immunohistochemistry. Additionally, IL-1ß, IL-4 and IL-8 concentrations in follicular fluid (FF) and serum were determined by enzyme-linked immunosorbent assay (ELISA). In granulosa and theca cells, IL-1RI, IL-1RII, IL-1RA and IL-4 expression levels were higher in cystic follicles than in the control dominant follicles. The serum and FF concentrations of IL-1ß and IL-4 showed no differences between groups, whereas IL-8 concentration was detected only in FF of cysts from cows with COD. The FF and serum concentrations of IL-1ß and IL-8 showed no significant differences, whereas IL-4 concentration was higher in FF than in serum in both the control and COD groups. These results evidenced an altered expression of cytokines in ovaries of cows with COD that could contribute to the pathogenesis of this disease.

Expression of TGFBR1, TGFBR2, TGFBR3, ACVR1B and ACVR2B is altered in ovaries of cows with cystic ovarian disease.

The objective of this study was to examine the expression of transforming growth factor beta receptor (TGFBR)1, TGFBR2, TGFBR3, activin receptor (ACVR)1B and ACVR2B in ovaries of cows with cystic ovarian disease (COD). The expression of the selected receptors was determined by immunohistochemistry in sections of ovaries from cows with ACTH-induced and spontaneous COD. Expression of TGFBR1 and TGFBR3 was higher in granulosa cells of cysts from cows with spontaneous COD than in tertiary follicles from the control group. Additionally, TGFBR3 expression was higher in granulosa cells of cysts from cows with ACTH-induced COD than in those from the control group and lower in theca cells of spontaneous and ACTH-induced cysts than in tertiary control follicles. There were no changes in the expression of TGFBR2. ACVR1B expression was higher in granulosa cells of tertiary follicles of cows with spontaneous COD than in the control group, whereas ACVR2B expression was higher in cysts of the spontaneous COD group than in tertiary follicles from the control group. The alterations here detected, together with the altered expression of the ligands previously reported, indicate alterations in the response of the ligands in the target cells, modifying their actions at cellular level.

Changes in the Proliferation/Apoptosis Balance in the Bovine Ovary: A Key Early Event in Follicular Persistence.

The objective of this work was to evaluate proliferation and apoptosis in the bovine ovary in a model of follicular persistence induced by low levels of progesterone to detect incipient changes during cystic ovarian disease development on the expected day of ovulation (day 0) and after 5, 10, and 15 days of follicular persistence. We analyzed cell proliferation by evaluating the expression of Ki-67 and apoptosis by evaluating caspase-3, BAX, and BCL2 expression. Proliferation was similar in the granulosa and theca cells of antral follicles in the P0 group (treated with progesterone up to the expected day of ovulation) and in the control group. A decrease in cell proliferation was detected after 5 days of persistence (P5) in relation to P0 (p < 0.05). Similar changes were found in the granulosa cells of the persistent follicles in relation to the control group (p < 0.05). Caspase-3 expression was similar in granulosa cells of antral follicles at early stages of persistence, with an increase after 15 days of persistence (p < 0.05). In the granulosa cells of group P10 (10 days of persistence), caspase-3 expression was reduced relative to that of antral follicles from the control group (p < 0.05). BCL2 expression was higher in granulosa cells of the persistent follicles of group P0 relative to the control follicles, with no changes in BAX expression, which was increased in persistent follicles of group P15 (p < 0.05). Similar results were observed in theca cells at initial stages of persistence. The results show that, initially, proliferation is maintained with low apoptosis and an increase in cell survival.

Follicular Cysts: A Single Sign and Different Diseases. A View from Comparative Medicine.

Ovarian cystic follicles are the sign of important causes of reproductive failure in numerous species. In this review, some morphological, endocrinological and clinical aspects of cystic follicles in women, cows, mares, sows and bitches are discussed. Follicular cysts are the consequence of the failure of a mature follicle to ovulate at the appointed time of ovulation in the estrous cycle. Although the etiology of follicular cysts remains unknown, this review examines the evidence about the role of endocrine signaling systems in the specific disease or syndrome in each of the species mentioned above. This review also describes, the changes in the pathways of endocrine mechanisms that would trigger disturbances in the intraovarian component underlying the aberrant persistence of follicular cysts. The knowledge of the morphological and endocrinological nature of cystic follicles in different species can provide relevant information to better understand specific diseases when it is integrally analyzed from the comparative medicine viewpoint.

BMP2, 4 and 6 and BMPR1B are altered from early stages of bovine cystic ovarian disease development.

Cystic ovarian disease (COD) is an important cause of subfertility in dairy cattle. Bone morphogenetic proteins (BMPs), mainly BMP2, BMP4 and BMP6, play a key role in female fertility. In this study, we hypothesized that an altered BMP system is associated with ovarian alterations contributing to COD pathogenesis. Therefore, we examined the expression of BMP2, BMP4 and BMP6 and BMP receptor 1B (BMPR1B) in the ovaries of animals with spontaneous or ACTH-induced COD, as well as during the development of the disease, in a model of follicular persistence induced by low doses of progesterone (at 5, 10 and 15 days of follicular persistence). Results showed changes in BMP2, BMP4 and BMP6 expression during folliculogenesis, in granulosa and theca cells in the COD groups, as well as at different stages of follicular persistence. Results also showed changes in BMPR1B expression in developing follicles in animals with COD, and at the initial stages of follicular persistence (P5). Comparison between groups showed significant differences, mainly in BMP4 and BMP6 expression, in granulosa and theca cells of different follicular categories. The expression of these BMPs also increased in cystic and persistent follicles, in relation to antral follicles of the control group. BMPR1B showed high expression in cystic follicles. Together, these results may indicate an alteration in BMPs, especially in BMP4 and BMP6, as well as in BMPR1B, which occurs early in folliculogenesis and incipiently during the development of COD, which could be a major cause of recurrence of this disease in cattle.Free Spanish abstract: A Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/early/2016/08/01/REP-15-0315/suppl/DC1.

Role of Glucocorticoids in Cystic Ovarian Disease: Expression of Glucocorticoid Receptor in the Bovine Ovary.

The aim of this study was to characterize the expression of glucocorticoid receptor (GR) in the components of normal bovine ovary and in animals with cystic ovarian disease (COD). Changes in the protein and mRNA expression levels were determined in control cows and cows with COD by immunohistochemistry and real-time PCR. GR protein expression in granulosa cells was higher in cysts from animals with spontaneous COD and adrenocorticotropic hormone-induced COD than in tertiary follicles from control animals. In theca interna cells, GR expression was higher in cysts from animals with spontaneous COD than in tertiary follicles from control animals. The increase in GR expression observed in cystic follicles suggests a mechanism of action for cortisol and its receptor through the activation/inactivation of specific transcription factors. These factors could be related to the pathogenesis of COD in cattle.

Characterization of persistent follicles induced by prolonged treatment with progesterone in dairy cows: an experimental model for the study of ovarian follicular cysts.

Cystic ovarian disease (COD) is a major factor contributing to poor reproductive efficiency of lactating dairy cows. The objective of the present study was to analyze the endocrine profile, growth dynamics, and histologic characteristics of persistent ovarian follicles-cysts developing in response to long-term administration of intermediate levels of progesterone. To this end, after synchronization of cows, a low dose of progesterone was administered for 5, 10, and 15 days after the expected day of ovulation in treated cows (groups P5, P10, and P15, respectively), using an intravaginal progesterone-releasing device. A significant increase in diameter was detected on Day 11 of progesterone treatment and thereafter (P < 0.05), and at Day 15 of persistence, the diameter of the persistent follicle reached a mean of 23 ± 0.6 mm. Microscopically, the persistent follicles had a complete granulosa, an intensely vascularized theca interna, and a collagenous theca externa layer. Temporal changes in the serum concentrations of estradiol, progesterone, and FSH were detected (effects of time, P < 0.01). Progesterone treatment completely inhibited the LH preovulatory surge in treated cows and affected the basal concentration of LH. The pulse frequency remained high at 5 and 10 days of persistence and declined (P < 0.05) after 15 days of persistence. The LH pulse concentration and pulse amplitude had a significant reduction (P < 0.05) during follicular persistence. Changes in the serum levels of estradiol, progesterone, 17-hydroxyprogesterone, and testosterone in serum and follicular fluid were also observed. In serum, estradiol increased gradually from proestrus to Day 10 of follicular persistence (P < 0.05), progesterone showed an increase (P < 0.05) at Day 5 of follicular persistence, 17-hydroxyprogesterone showed a significant decrease at 5 days of follicular persistence in relation to proestrus, and testosterone showed a significant increase (P < 0.05) from proestrus and Day 5 of persistence through Day 15 of follicular persistence. Correlation between serum and follicular fluid steroid concentrations was significant for testosterone (P < 0.0001) and not significant for estradiol and progesterone. These findings indicate that ovarian cysts in COD are similar in many ways to the persistent follicles induced by progesterone, with an analogous hormonal and morphologic context, thus confirming a local role of subluteal levels of progesterone in COD pathogenesis and in the regulatory mechanisms of the ovarian function.

Neonatal androgenization-induced early endocrine-metabolic and ovary misprogramming in the female rat.

AIM: Androgen excess predisposes the organism to develop metabolic-endocrine and reproductive dysfunctions, among them the development of a phenotype resembling that of human Polycystic Ovary Syndrome (PCOS). METHODS: We analyzed the impact of a single neonatal (5day-old) testosterone propionate (TP; s.c. 1.25mg/female pup) dose on: a) several metabolic-endocrine activities and b) ovarian steroidogenic and granulosa cell (GC) functions and also follicular population in juvenile and adult TP and control (CT) rats. KEY FINDINGS: Compared to CT rats, TP animals were characterized by: a) accelerated growth, hyperadiposity and hyperleptinemia, b) very early (pre-weaning age) vaginal opening, c) hyperinsulinemia in adult life, d) dysfunctional ovarian steroidogenesis, e) conserved GC functionality in both juveniles (in vitro) and adults (in vivo), and f) estrous cycles arrested at estrus. Finally, histological studies of the ovaries indicated that in TP (vs. CT) rats: i) primary and antral follicle frequencies were 3- and 15-fold higher and lower, respectively, in juveniles and ii) secondary and atretic follicle frequencies were 3- and 5-fold lower and higher, respectively, in adults. Large cystic images without corpus luteum were observed in the ovaries from adult TP rats only. SIGNIFICANCE: Our results strongly suggest that transient neonatal hyperandrogenemia induced early misprogramming of metabolic-endocrine and ovarian (steroidogenesis/folliculogenesis) functions. Conversely, TP rats preserved their ovary GC endocrine function. Our results further support the high risk of developing ovarian hyperstimulation syndrome for infertile women with transient/chronic hyperandrogenemia (PCOS) subjected to assisted reproductive technologies.

Molecular aspects of bovine cystic ovarian disease pathogenesis.

Cystic ovarian disease (COD) is one of the main causes of reproductive failure in cattle and causes severe economic loss to the dairy farm industry because it increases both days open in the post partum period and replacement rates due to infertility. This disease is the consequence of the failure of a mature follicle to ovulate at the time of ovulation in the estrous cycle. This review examines the evidence for the role of altered steroid and gonadotropin signaling systems and the proliferation/apoptosis balance in the ovary with cystic structures. This evidence suggests that changes in the expression of ovarian molecular components associated with these cellular mechanisms could play a fundamental role in the pathogenesis of COD. The evidence also shows that gonadotropin receptor expression in bovine cystic follicles is altered, which suggests that changes in the signaling system of gonadotropins could play a fundamental role in the pathogenesis of conditions characterized by altered ovulation, such as COD. Ovaries from animals with COD exhibit a disrupted steroid receptor pattern with modifications in the expression of coregulatory proteins. These changes in the pathways of endocrine action would trigger the changes in proliferation and apoptosis underlying the aberrant persistence of follicular cysts. Free Spanish abstract: A Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/149/6/R251/suppl/DC1.

mRNA expression pattern of gonadotropin receptors in bovine follicular cysts.

Follicular growth and steroidogenesis are dependent on gonadotropin binding to their receptors in granulosa and theca cells of ovarian follicles. The aim of the present study was to evaluate the expression patterns of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHCGR) in ovarian follicular structures from cows with cystic ovarian disease (COD) as compared with those of regularly cycling cows. Relative real-time RT-PCR analysis showed that the expression of FSHR mRNA in granulosa cells was highest in small antral follicles, then decreased significantly as follicles increased in size, and was lowest in cysts. FSHR mRNA was not detected in the theca cells of any follicular category, including cysts. LHCGR mRNA expression in granulosa cells was significantly higher in large antral follicles than in cysts, and not detected in granulosa cells of small and medium antral follicles. In theca cells, the expression level of LHCGR mRNA in medium antral follicles was higher than in small and large antral follicles, whereas that in follicular cysts it was similar to those in small and medium antral follicles, but higher than that in large antral follicles. Our findings provide evidence that there is an altered gonadotropin receptor expression in bovine cystic follicles, and suggest that in conditions characterized by altered ovulation, such as COD, changes in the signaling system of gonadotropins may play a fundamental role in their pathogenesis.

Developmental programming: prenatal steroid excess disrupts key members of intraovarian steroidogenic pathway in sheep.

Prenatal testosterone (T) excess disrupts ovarian cyclicity and increases circulating estradiol levels as well as follicular recruitment and persistence culminating in multifollicular ovary similar to women with polycystic ovary syndrome. We tested whether prenatal T excess, by androgenic or estrogenic action, disrupts the steroid biosynthetic machinery in sheep in a cell-, follicle stage-, age-, and treatment-specific manner consistent with the ovarian disruptions and increased estradiol release. Impact of T/dihydrotestosterone (DHT) treatments from days 30-90 of gestation on steroidogenic acute regulatory protein, 3ß-hydroxysteroid dehydrogenase, cytochrome P-450 17α-hydroxylase/C17, 20-lyase (CYP17A1), and cytochrome P-450 aromatase (CYP19A1) were examined on fetal day 90, 140 and 10 months (postpubertal), and 21 months (adult, no DHT group) of age by immunohistochemistry. All 4 markers changed in a cell-, follicle stage-, and age-specific manner. Both treatments increased steroidogenic acute regulatory protein expression in preantral follicles of postpubertal and adult females. Effects of prenatal T and DHT on 3ß-hydroxysteroid dehydrogenase differed in a follicle- and age-specific manner. CYP17A1 was reduced in the theca interna of antral follicles by T, but not DHT, in 10- and 21-month-old females. CYP19A1 was reduced by both T and DHT at all ages barring an increase on fetal day 140. Reduced granulosa CYP19A1 and thecal CYP17A1 in adults likely disrupt the intrafollicular androgen/estrogen balance contributing to follicular persistence. The reduced thecal CYP17A1 expression suggests that the hyperandrogenic ovarian phenotype may originate from increased enzyme activity or alternatively via a different isoform of CYP17. The reduced CYP19A1 in antral follicles of adults indicates that the increased circulating estradiol release likely arises from the increased number of persisting follicles.

Role of activin, inhibin, and follistatin in the pathogenesis of bovine cystic ovarian disease.

Cystic ovarian disease (COD) is an important cause of infertility in dairy cattle. Although many researchers have focused their work on the endocrine changes related to this disease, evidence indicates that intraovarian components play an important role in follicular persistence. Activin, inhibin, and follistatin participate as intraovarian regulatory molecules involved in follicular cell proliferation, differentiation, steroidogenesis, oocyte maturation, and corpus luteum function. Given the importance of these factors in folliculogenesis, we examined the expression and immunolocalization of activin/inhibin ßA-subunit, inhibin α-subunit, and follistatin in the ovaries of healthy estrus-synchronized cows and in those of cows with spontaneous or adrenocorticotropic hormone (ACTH)-induced COD. We also studied inhibin B (α ßB) levels in serum and follicular fluid. We found an increased expression of the ßA-subunit of activin A/inhibin A, the α-subunit of inhibin, and follistatin in granulosa cells of spontaneous follicular cysts by immunohistochemistry, and decreased concentrations of inhibin B (α ßB) in the follicular fluid of spontaneous follicular cysts. These results, together with those previously obtained, indicate that the expression of the components of the activin-inhibin-follistatin system is altered. This could lead to multiple alterations in important functions in the ovary like the balance between pro- and anti-apoptotic factors, follicular proliferation/apoptosis, and steroidogenesis, which may contribute to the follicular persistence and endocrine changes found in cattle with COD.