Effects of active immunization against GnRH on serum LH, inhibin A, sexual development and growth rate in Chinese female pigs.

Surgical castration of young female pigs is common practice in Chinese pig farming today. The purpose of the present study is to investigate anti-GnRH immunization as a practical alternative to surgical castration for female pigs. Thirty-six Chinese female crossbred pigs (Chinese Yanan x Yorkshire) were selected from 12 litters, three pigs from each litter, at the age of 10-13 weeks. One pig from each litter was immunized with 62.5 microg D-Lys6-GnRH-tandem-dimer peptide conjugated to ovalbumin in Specol adjuvant at Week 0 (0 week post-vaccination, wpv), and a booster vaccination was given 8 weeks later (8 wpv). Its intact and castrate littermates (surgically castrated at the time of weaning, i.e. at 6 weeks of age) were administered the vehicle and served as controls. Antibody titers, serum LH and inhibin A were determined at the day of first vaccination, every 4 weeks thereafter and at the day of slaughter (18 wpv). At slaughter, ovaries were inspected for the presence of follicles and corpora lutea, and ovarian and uterine weights were recorded. Ten of twelve immunized pigs responded well to the immunization (immunocastrated animals), while the remaining two pigs responded poorly (nonresponders). Antibody titres in immunocastrated animals steadily increased after immunization, became maximal at 12 wpv and remained high until slaughter. Serum LH levels were reduced (P < 0.05) in immunocastrated pigs as compared to intact controls and surgical castrates. Serum inhibin A levels decreased after vaccination, and equaled surgical castrate levels from 8 wpv until the end of the experiment. Ovarian and uterine weights (1.3 +/- 0.2 and 43.9 +/- 11.4 g, respectively; mean +/- S.E.M.) were significantly lower (P < 0.05) in immunocastrates than in intact controls (9.4 +/- 1.1 and 390.9 +/- 67.2 g, respectively). Antibody titers were significantly lower (P < 0.05) in nonresponders than in immunocastrated pigs from 12 wpv to slaughter. Ovarian and uterine weights were similar in nonresponders and in intact controls. Macroscopically, no follicular structures were found in ovaries of immunocastrated pigs, while large follicles or corpora lutea were observed in the ovaries of both nonresponders and intact controls. Although not significant, immunocastrates had a numerically higher average daily gain than surgical castrates and intact controls (0.74 +/- 0.04 versus 0.66 +/- 0.04 versus 0.66 +/- 0.03 kg per day, respectively; mean +/- S.E.M., P = 0.09). Results obtained in the present study demonstrate that anti-GnRH immunization can be an attractive alternative to surgical castration for Chinese crossbred female pigs. Our results also question the beneficial effect of surgical castration on growth as compared to intact controls.

Inhibins/activins as diagnostic markers for ovarian cancer.

It is widely recognised that the early detection and subsequent assessment of recurrence of ovarian cancers are key steps for successful treatment. Available serum markers (e.g. CA125) are sensitive for some epithelial carcinomas (e.g. serous, endometrioid, clear cell), however, these markers are less sensitive for granulosa cell tumours and mucinous carcinomas. Serum inhibin is an ovarian product which decreases to non detectable levels after menopause, however, certain ovarian cancers (mucinous carcinomas and sex cord stromal tumours such as granulosa cell tumours) continue to produce inhibin which provides a basis for a serum diagnostic test. Studies from this and other laboratories have investigated the suitability of inhibin as a diagnostic marker by identifying which inhibin (inhibin A (alphabetaA), inhibin B (alphabetaB), free alpha subunit) or activin (betaAbetaA) form is associated with these cancers. Available data show that inhibin assays which detect all inhibin forms, i.e. assays which detect the alpha subunit both as the free form and as an alphabeta subunit dimer provide the highest sensitivity/specificity characteristics as an ovarian cancer diagnostic test. This review will discuss the data supporting these observations and show recent studies in which a new alpha subunit monoclonal antibody-based ELISA is used as a potential diagnostic test. Furthermore, based on the high sensitivity/specificity characteristics of the respective assays for the various types of ovarian cancer, the combination of the inhibin assay with CA125 detects the majority of all ovarian cancers.

Plasma FSH, inhibin A and inhibin isoforms containing pro- and -alphaC during winter anoestrus, spring transition and the breeding season in mares.

Ten mares were studied from February (winter anoestrus) to their second ovulation in the breeding season to investigate the relationship between resumption of ovarian cyclicity in the spring and circulating concentrations of FSH, inhibin A and inhibin isoforms containing pro- and -alphaC immunoreactivity. An additional four mares were studied during one oestrous cycle. Growth and regression of ovarian follicles were monitored by transrectal ultrasonography. The frequency of blood sampling varied from three times a week to once a day, depending on the follicular activity present. Concentrations of FSH, oestradiol, inhibin A and pro- and -alphaC isoforms were low during deep winter anoestrus when minimal follicular activity was present in the ovaries. During spring transition, an increase in FSH concentration preceded the emergence of each follicular wave. Concentrations of inhibins were significantly higher (P < 0.05) during growth of anovulatory follicles in spring transition than during winter anoestrus. Plasma concentrations of oestradiol and inhibin A were significantly higher (P < 0.001, P < 0.05, respectively) during the growth of preovulatory follicles than during the growth of transitional anovulatory follicles, but concentrations of inhibin pro-alphaC isoforms did not differ between the two types of follicle. During the oestrous cycle, there was a significant inverse relationship (P < 0.001) between concentrations of FSH and the inhibins. Plasma inhibin pro-alphaC isoforms, but not inhibin A, reached a peak on the day of ovulation. The results strongly indicate that FSH regulates growth of spring anovulatory and preovulatory follicles. Inhibins are likely to contribute to negative feedback on the release of FSH from the pituitary gland both during the transitional period and the breeding season in mares.

Characterization of inhibin forms and their measurement by an inhibin alpha-subunit ELISA in serum from postmenopausal women with ovarian cancer.

The aim of this study was to characterize the molecular wt forms of inhibins A and B and its free alpha-subunit present in serum from women with ovarian cancer as a basis for developing improved monoclonal antibody-based inhibin assays for monitoring ovarian cancer. Three new inhibin alpha-subunit (alphaC) ELISAs were developed using monoclonal antibodies directed to three nonoverlapping peptide regions of the alphaC region of the inhibin alpha-subunit. To characterize serum inhibin molecular wt forms present in women with ovarian cancer, existing inhibin immunoassays (inhibin A, inhibin B, and pro-alphaC) and the new alphaC ELISAs were applied to sera from women with granulosa cell tumors and mucinous carcinomas previously fractionated using a combined immunoaffinity chromatography, preparative SDS-PAGE, and electroelution procedure. The distribution and molecular size of dimeric inhibins and alpha-subunit detected were consistent with known mol wt forms of inhibins A and B and inhibin alpha-subunit and their precursor forms present in serum and follicular fluid from healthy women. The alphaC ELISAs recognized all known forms of inhibin and the free inhibin alpha-subunit, although differences between alphaC ELISAs were observed in their ability to detect high mol wt forms. To assess which of the alphaC ELISAs was preferred in application to ovarian cancer, the alphaC ELISAs were applied to serum from a range of normal postmenopausal women (n = 61) and postmenopausal women (n = 152) with ovarian (serous, mucinous, endometrioid, clear cell carcinomas, and granulosa cell tumors) and nonovarian (breast and colon) cancers. Despite differences in their ability to detect high mol wt forms of inhibin, the alphaC ELISAs showed similar sensitivity (i.e. proportion of cancer patients correctly detected) and specificity (proportion of controls correctly detected) indexes in the detection of mucinous carcinomas (84% and 95%) and granulosa cell tumors (100% and 95%) compared with earlier inhibin RIA or polyclonal antibody-based immunofluorometric assays. A combination of the alphaC ELISAs with the CA125 assay, an ovarian tumor marker that has a high sensitivity and specificity for other ovarian cancers (serous, clear cell, and endometrioid), resulted in an increase in sensitivity/specificity indexes (95% and 95%) for the all ovarian cancer group. These new monoclonal antibody-based inhibin alphaC ELISAs now provide practical and sensitive assays suitable for evaluation as diagnostic tests for monitoring ovarian cancers.

Development of an inhibin alpha subunit ELISA with broad specificity.

Inhibin immunoassays with a sufficiently broad specificity to detect all alpha subunit-containing forms are of value in detecting and monitoring various ovarian cancers. Assays to date with this specificity are not readily amenable to wide diagnostic application. The objective of this study was to develop a sensitive two-site ELISA using alpha subunit-directed monoclonal antibodies (Mabs) able to detect all forms of inhibin to replace a previously described alpha subunit-directed immunofluorometric assay (IFMA). In this study, the major inhibin epitopes in the two polyclonal antisera used in the alphaC IFMA were initially identified and Mabs were raised to these regions. These Mabs in conjunction with the inhibin alpha subunit R1 Mab (Groome) were used to develop alpha subunit ELISAs with high sensitivity. Application of these assays to human serum and human follicular fluid following fractionation by an immunoaffinity/preparative PAGE/electroelution procedure which separated inhibins according to their molecular weights, indicated that the specificity of the various ELISAs differed between Mab combinations with preferences noted for either the alpha subunit or dimeric forms. A combination of Mabs in an ELISA was identified which provided data which matched that obtained with the alphaC IFMA and which may be useful as a replacement inhibin assay in clinical studies.