In avian species, the granulosa cells produce progesterone (P), but not testosterone (T) or estradiol (E). The theca folliculi in avian species produces T and E and is anatomically comprised of the theca interna and the theca externa. It is not known, however, whether both T and E are produced by the same cell type. In the present study, preovulatory follicles of adult female turkeys were separated into their anatomical subdivisions, the granulosa, theca interna, and theca externa, and the relative steroidogenic contribution by each layer was investigated. Granulosa cells from the largest (F1) and fifth largest (F5) preovulatory follicles produced increasing levels of P in response to ovine LH (oLH; 0-10 micrograms/ml); however, T and E were not detectable. Theca interna and theca externa cells from F5 follicles were cultured alone or in combination. Theca interna cells produced P and T, but not E. Theca externa cells produced P, T, and E. E production was greatly enhanced by coincubation of cells from the two theca layers. oLH, (0-100 ng/ml) stimulated theca interna P and T production and E production by the coincubation of theca interna and theca externa cells. When theca interna and theca externa cells from the F1 and F5 follicles were tested, theca externa cells from F5, but not F1, converted androstenedione and T, but not P into E, indicating a loss of aromatase activity in the theca externa with follicular maturation. Neither F1 nor F5 theca interna cells converted P, A, or T into estradiol, providing additional evidence that aromatase activity is limited exclusively to the theca externa. Addition of oLH (0-100 ng/ml) did not increase conversion of T into E by the F5 theca externa cells, suggesting that production of E by the theca externa is substrate driven and not influenced by direct LH action. Avian ovarian steroidogenesis is described using a three-cell model, with the principle sources of P, T, and E being the granulosa, theca interna, and theca externa cells, respectively.

Intracranial perfusion of ovine prolactin (oPrl) via osmotic pump in laying turkey hens caused a sudden onset in incubation behavior, defined as an increase in nest visits. The hens also displayed a gradual decrease in egg laying during the time they were receiving oPrl, another indicator of the onset of incubation. Circulating immunoreactive turkey Prl levels fell during the perfusion period, even though the hens were displaying persistent nesting activity and reduced egg laying. No effects on serum LH were noted. Perfusion of oPrl during the first 14 days of photostimulation delayed the onset of egg laying by several days. No effects on serum Prl or serum LH were noted. It is suggested that incubation behavior is facilitated by central levels of Prl.

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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.

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Although vasoactive intestinal peptide (VIP) is a well characterized physiological PRL-releasing factor in avian species, its regulated expression is not fully understood. We cloned complementary DNAs encoding the prepro-turkey VIP (prepro-tVIP) molecule from an adult turkey hypothalamic complementary DNA library. When the amino acid sequence of the prepro-tVIP was compared to chicken and mammalian sequence, it was found that the isolated tVIP molecules lacked the 27-amino acid peptide histidine isoleucine (PHI) portion of the precursor protein. Several tissues showed an alternatively spliced tVIP transcript that lacked the PHI sequence. Only in the hypothalamus did tVIP-specific primer pairs and reverse transcription-polymerase chain reaction produce two alternatively spliced fragments. The larger hypothalamus-specific fragment was subjected to nucleotide sequence analysis and identified as containing the alternatively spliced PHI-containing exon, which encoded a 27-amino acid PHI peptide in addition to the 8 amino acids that flanked the peptide. Hypothalamic tVIP expression was shown to be up-regulated during the incubation phase of the reproductive cycle. The increased steady state level of tVIP messenger RNA appears to be regulated by nesting behavior, because nest deprivation dramatically suppressed its expression. Levels of the minor tVIP transcript containing both the PHI- and VIP-encoding exons did not significantly change between reproductive stages and were maintained at approximately 4-6% of the total tVIP transcript level. Our findings provide further evidence that VIP is the most important PRL-releasing factor in birds. Our study should serve as a useful model for determining whether PHI contributes in any way to the physiological role of PRL regulation. Revealing the tissue distribution of VIP and PHI gene expression and tissue-specific alternative splicing could contribute to an understanding of the physiological functions of the two peptides as well as their relative roles in PRL regulation.