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The ovary is a complex organ composed of cells of diverse lineages. Therefore, in this study we examined whether immunolocalization of various cytoskeletal, epithelial, immune-cell, and neural-associated proteins can differentiate various cells in the baboon and human ovaries. Surface epithelial cells exhibited immunoreactivity for cytokeratin and desmin, however, they did not immunostain for other epithelial markers such as carcinoembryonic antigen or epithelial membrane antigen. Smooth muscle actin was distributed apically whereas vimentin was localized basally in these cells. Ova exhibited strong immunoreactivity for S-100, Leu-M1, and neurofilament and did not show immunoreactivity for epithelial and cytoskeletal proteins. In antral follicles and theca cells, and after formation of corpus luteum, both granulosa and theca cells expressed immunoreactivity for vimentin. Cytokeratins were absent in the preantral and antral follicles. However, atresia and development of apoptosis was associated with expression of immunoreactive cytokeratins in atretic follicles. Development of corpus luteum led to major changes in the immunophenotype of follicular cells. The mere presence of immunoreactivity for cytokeratin and a strong immunoreactivity for desmin in the luteinized granulosa and not in the theca cells allowed discrimination of these cells from each other and from their ancestral cells. Proliferating cell nuclear antigen was present in various ovarian cells except for ovum. The distinct patterns of expression of cytoskeletal, epithelial, and neural-associated proteins in various cells of the ovary facilitates their identification and discrimination.

The effect of exogenous ovine prolactin (oPRL) on preovulatory follicle P450 17 alpha-hydroxylase (C17) and aromatase (ARO) mRNA abundance was investigated in turkeys. Ovine PRL (124 IU/hen per day) was injected i.m. into four sets (n = 8) of laying turkeys for 2, 4, 8, or 14 days. Vehicle was injected into control hens for 8 days (n = 8). Blood samples were collected and serum was assayed for LH, progesterone (P), testosterone (T), and estradiol (E). Theca layers from the largest (F1) and the third (F3), fifth (F5), and seventh (F7) largest preovulatory follicles and from small white follicles (SWF) were examined for C17 and ARO mRNA contents. The number of atretic follicles increased from 0 (vehicle-injected controls) to 9 (14-day-oPRL-injected hens). Serum E, T, and LH levels decreased, while P levels remained unchanged. There was a transient increase in theca C17 mRNA abundance of 2- and 4-day-oPRL-treated hen follicles. Cytochrome P450 ARO mRNA levels were reduced in SWF and F7 in response to oPRL. Thecal C17 and ARO mRNA content was reduced during follicular maturation in laying hens. ARO mRNA was not detectable in granulosa cells. The progressive decline in C17 and ARO mRNA content associated with follicular maturation as well as the absence of ARO mRNA in granulosa cells is consistent with the secretory activity of P, T, and E in preovulatory follicles. These findings suggest that reduced circulating E may be a consequence of suppressed ARO gene expression whereas the oPRL suppression of T secretion may not be coupled to C17 gene expression.

This study was designed to examine changes in cytochrome P450 17a-hydroxylase (C17) and aromatase (ARO) mRNA contents in the theca layer of preovulatory follicles (POF) as turkey hens transit from egg laying to incubation. Hens were grouped into the following categories: 1) laying hens-laid one egg per day and nested 1-2 times per day; 2) transitional hens-laid one egg per day and nested > 4 times per day; and 3) Day 1, Day 3, and Day 5 incubating hens-laid no eggs for 2, 4, or 6 days, respectively, and nested > 4 times per day. Small white follicles (SWF) and the theca layer from the largest (F.) and the third (F3), fifth (F 5), and seventh (F 7 ) largest POF were dispersed and challenged with testosterone (T) for 5 h. Relative levels of C17 and ARO mRNA were examined from the theca layers of F 1, F3, F, F 7, and SWF. The number of atretic follicles increased from O (layers) to 8 (Day 5 incubating hens). Serum LH, progesterone (P), and estradiol (E), but not T, declined on Day 1 of incubation. Basal levels of P, T, and E from theca and SWF cells declined in incubating hens. Both basal and T-stimulated theca and SWF production of E decreased in incubating hens. C17 and ARO mRNA declined in SWF, F 7, and F5 during follicular atresia. It is suggested that reduced gene expression of ovarian steroidogenic enzymes may be a partial determinant of reduced circulating sex steroid levels in incubating hens. Follicular maturation is associated with a progressive decrease in C17 and ARO mRNA steady state levels as the follicle matures; this may partially explain the decreases in T and E secretion that are associated with follicular maturation.

An inverse relationship often exists between prolactin (Prl) and LH in avian species. Our study was designed to investigate the relationship between hypothalamic and posterior pituitary vasoactive intestinal peptide (VIP)--an endogenous Prl-releasing peptide--and chicken GnRH-I and GnRH-II. Hypothalamic VIP content was increased after photostimulation, reaching its highest levels in incubating and photorefractory birds. The highest hypothalamic GnRH-I content was in laying hens followed by that in photostimulated and incubating birds. The lowest levels were in the nonphotostimulated birds. Hypothalamic GnRH-II increased after photostimulation, then fell to nonphotostimulated levels during incubation and photorefractoriness. Posterior pituitary VIP content was elevated in response to photostimulation, reaching a peak value in the laying and incubating birds, then declining in the photorefractory hens. Posterior pituitary GnRH-I and GnRH-II content peaked in the incubating birds. Ovariectomy caused a significant reduction in hypothalamic GnRH-I and GnRH-II with no effect on VIP; no changes were found in the posterior pituitary due to ovariectomy. Reducing day length caused a significant decrease in the hypothalamic and the posterior pituitary content of VIP and GnRH-I, and GnRH-II. Ovine Prl (oPrl) administration to laying hens reduced the hypothalamic VIP and GnRH-I and GnRh-II content. Posterior pituitary GnRH-I content was also reduced. Although GnRH-II levels were reduced by Day 4 of injections, they rose to peak levels by Day 14 of oPrl administration. Posterior pituitary VIP content was not altered by oPrl.

The effect of ovine prolactin (oPRL) administration to laying and incubating turkey hens was studied. In experiment one, five treatment groups (n = 6) of laying hens received injections of vehicle or oPRL (4 mg/bird/day) for 2, 4, 8, or 14 days. Hypothalamic vasoactive intestinal peptide (VIP) content decreased to its lowest level (p < 0.05) by Day 2 of oPRL injection. Serum turkey PRL decreased by Day 8 of injection, reaching nadir by Day 14. Similar to the decline in hypothalamic VIP content, the maximal decline in the number of anterior pituitary VIP binding sites was achieved by Day 2 of oPRL administration. Hypothalamic GnRH-I content decreased after 2 days of oPRL injection and remained low through Day 14 of the experiment. GnRH-II levels declined with time, reaching significantly lower levels after 8 days of injections, and remained low through Day 14. Plasma LH levels also declined (p < 0.05) after Day 14 of oPRL administration. In experiment 2, two groups (n = 6) of incubating birds were used: controls receiving injections of vehicle only and birds receiving injections of oPRL (4 mg/bird/day) for 10 days. Exogenous oPRL had no effect on hypothalamic VIP, GnRH-I or II, anterior pituitary VIP binding sites, or plasma turkey PRL or LH. The findings suggest that in laying hens, PRL inhibits its own secretion by acting on both the hypothalamus and the anterior pituitary; this phenomenon does not occur in incubating birds. Furthermore, we provide evidence that pRL acts centrally to reduce LH levels by reducing GnRH levels in the hypothalamus.

Although previous studies have shown that the tumor necrosis factor-alpha (TNF) gene is expressed in pregnancy decidua, it has not been determined whether this gene is transcribed or translated in the endometrium prior to implantation. To address this question and to identify cells expressing the TNF gene, samples of normal cycling human endometria were tested for TNF mRNA by in situ hybridization using a biotinylated antisense RNA probe, and the corresponding protein was localized in the same tissues by immunocytochemistry with a monoclonal antibody to TNF. Both TNF message and protein were identified in the endometrium throughout the menstrual cycle, and the same types of cells that contained transcripts also contained protein. As judged by the intensities of the hybridization signals, TNF mRNA increased during the proliferative phases, declined in the early secretory phase, and again rose during mid-to-late secretory phases, suggesting positive associations with levels of female sex hormones that show similar cyclic fluctuations. At the initiation of the cycle, transcripts were primarily localized to glandular epithelial cells whereas by the midproliferative phase, message was also present in stromal cells. Hybridization signals were consistently more intense in functionalis-region than in basalis-region stromal cells, and were frequently stronger in basalis-region glandular epithelia than in functionalis-region glands. These observations document that the TNF gene is expressed in normal cycling endometria, suggest that ovarian hormones may regulate TNF gene transcription, and identify differences in specific endometrial compartments. The findings are also consistent with the postulate that TNF is a local mediator of cellular communications in the human endometrium.

The effect of exogenous ovine prolactin (oPRL) on preovulatory follicle P450 17 alpha-hydroxylase (C17) and aromatase (ARO) mRNA abundance was investigated in turkeys. Ovine PRL (124 IU/hen per day) was injected i.m. into four sets (n = 8) of laying turkeys for 2, 4, 8, or 14 days. Vehicle was injected into control hens for 8 days (n = 8). Blood samples were collected and serum was assayed for LH, progesterone (P), testosterone (T), and estradiol (E). Theca layers from the largest (F1) and the third (F3), fifth (F5), and seventh (F7) largest preovulatory follicles and from small white follicles (SWF) were examined for C17 and ARO mRNA contents. The number of atretic follicles increased from 0 (vehicle-injected controls) to 9 (14-day-oPRL-injected hens). Serum E, T, and LH levels decreased, while P levels remained unchanged. There was a transient increase in theca C17 mRNA abundance of 2- and 4-day-oPRL-treated hen follicles. Cytochrome P450 ARO mRNA levels were reduced in SWF and F7 in response to oPRL. Thecal C17 and ARO mRNA content was reduced during follicular maturation in laying hens. ARO mRNA was not detectable in granulosa cells. The progressive decline in C17 and ARO mRNA content associated with follicular maturation as well as the absence of ARO mRNA in granulosa cells is consistent with the secretory activity of P, T, and E in preovulatory follicles. These findings suggest that reduced circulating E may be a consequence of suppressed ARO gene expression whereas the oPRL suppression of T secretion may not be coupled to C17 gene expression.

This study was designed to examine changes in cytochrome P450 17 alpha-hydroxylase (C17) and aromatase (ARO) mRNA contents in the theca layer of preovulatory follicles (POF) as turkey hens transit from egg laying to incubation. Hens were grouped into the following categories: 1) laying hens--laid one egg per day and nested 1-2 times per day; 2) transitional hens--laid one egg per day and nested4 times per day; and 3) Day 1, Day 3, and Day 5 incubating hens--laid no eggs for 2, 4, or 6 days, respectively, and nested4 times per day. Small white follicles (SWF) and the theca layer from the largest (F1) and the third (F3), fifth (F5), and seventh (F7) largest POF were dispersed and challenged with testosterone (T) for 5 h. Relative levels of C17 and ARO mRNA were examined from the theca layers of F1, F3, F5, F7, and SWF. The number of atretic follicles increased from 0 (layers) to 8 (Day 5 incubating hens). Serum LH, progesterone (P), and estradiol (E), but not T, declined on Day 1 of incubation. Basal levels of P, T, and E from theca and SWF cells declined in incubating hens. Both basal and T-stimulated theca and SWF production of E decreased in incubating hens. C17 and ARO mRNA declined in SWF, F7, and F5 during follicular atresia. It is suggested that reduced gene expression of ovarian steroidogenic enzymes may be a partial determinant of reduced circulating sex steroid levels in incubating hens.(ABSTRACT TRUNCATED AT 250 WORDS)

An inverse relationship often exists between prolactin (Prl) and LH in avian species. Our study was designed to investigate the relationship between hypothalamic and posterior pituitary vasoactive intestinal peptide (VIP)--an endogenous Prl-releasing peptide--and chicken GnRH-I and GnRH-II. Hypothalamic VIP content was increased after photostimulation, reaching its highest levels in incubating and photorefractory birds. The highest hypothalamic GnRH-I content was in laying hens followed by that in photostimulated and incubating birds. The lowest levels were in the nonphotostimulated birds. Hypothalamic GnRH-II increased after photostimulation, then fell to nonphotostimulated levels during incubation and photorefractoriness. Posterior pituitary VIP content was elevated in response to photostimulation, reaching a peak value in the laying and incubating birds, then declining in the photorefractory hens. Posterior pituitary GnRH-I and GnRH-II content peaked in the incubating birds. Ovariectomy caused a significant reduction in hypothalamic GnRH-I and GnRH-II with no effect on VIP; no changes were found in the posterior pituitary due to ovariectomy. Reducing day length caused a significant decrease in the hypothalamic and the posterior pituitary content of VIP and GnRH-I, and GnRH-II. Ovine Prl (oPrl) administration to laying hens reduced the hypothalamic VIP and GnRH-I and GnRh-II content. Posterior pituitary GnRH-I content was also reduced. Although GnRH-II levels were reduced by Day 4 of injections, they rose to peak levels by Day 14 of oPrl administration. Posterior pituitary VIP content was not altered by oPrl.

The effect of ovine prolactin (oPRL) administration to laying and incubating turkey hens was studied. In experiment one, five treatment groups (n = 6) of laying hens received injections of vehicle or oPRL (4 mg/bird/day) for 2, 4, 8, or 14 days. Hypothalamic vasoactive intestinal peptide (VIP) content decreased to its lowest level (p0.05) by Day 2 of oPRL injection. Serum turkey PRL decreased by Day 8 of injection, reaching nadir by Day 14. Similar to the decline in hypothalamic VIP content, the maximal decline in the number of anterior pituitary VIP binding sites was achieved by Day 2 of oPRL administration. Hypothalamic GnRH-I content decreased after 2 days of oPRL injection and remained low through Day 14 of the experiment. GnRH-II levels declined with time, reaching significantly lower levels after 8 days of injections, and remained low through Day 14. Plasma LH levels also declined (p0.05) after Day 14 of oPRL administration. In experiment 2, two groups (n = 6) of incubating birds were used: controls receiving injections of vehicle only and birds receiving injections of oPRL (4 mg/bird/day) for 10 days. Exogenous oPRL had no effect on hypothalamic VIP, GnRH-I or II, anterior pituitary VIP binding sites, or plasma turkey PRL or LH. The findings suggest that in laying hens, PRL inhibits its own secretion by acting on both the hypothalamus and the anterior pituitary; this phenomenon does not occur in incubating birds. Furthermore, we provide evidence that pRL acts centrally to reduce LH levels by reducing GnRH levels in the hypothalamus.

Although previous studies have shown that the tumor necrosis factor-alpha (TNF) gene is expressed in pregnancy decidua, it has not been determined whether this gene is transcribed or translated in the endometrium prior to implantation. To address this question and to identify cells expressing the TNF gene, samples of normal cycling human endometria were tested for TNF mRNA by in situ hybridization using a biotinylated antisense RNA probe, and the corresponding protein was localized in the same tissues by immunocytochemistry with a monoclonal antibody to TNF. Both TNF message and protein were identified in the endometrium throughout the menstrual cycle, and the same types of cells that contained transcripts also contained protein. As judged by the intensities of the hybridization signals, TNF mRNA increased during the proliferative phases, declined in the early secretory phase, and again rose during mid-to-late secretory phases, suggesting positive associations with levels of female sex hormones that show similar cyclic fluctuations. At the initiation of the cycle, transcripts were primarily localized to glandular epithelial cells whereas by the midproliferative phase, message was also present in stromal cells. Hybridization signals were consistently more intense in functionalis-region than in basalis-region stromal cells, and were frequently stronger in basalis-region glandular epithelia than in functionalis-region glands. These observations document that the TNF gene is expressed in normal cycling endometria, suggest that ovarian hormones may regulate TNF gene transcription, and identify differences in specific endometrial compartments. The findings are also consistent with the postulate that TNF is a local mediator of cellular communications in the human endometrium.