A. J. Conley, I. M. Bird; The Role of Cytochrome P450 17α-Hydroxylase and 3β-Hydroxysteroid Dehydrogenase in the Integration of Gonadal and Adrenal Steroidogen

Two groups of 10 Holstein cows were chosen by pairs from a 20-yr genetic selection project that used either breed average or breed high sires chosen only for Predicted Differences in milk production. Milk production (305-d mature equivalent) was 10,814 kg and 6912 kg for the high and average groups of cows. Days to first visual estrus and number of ovulations before first visual estrus were greater for the high versus the average group (66 vs. 43 d and 1.6 vs. .7 ovulations). No differences were significant between groups for the interval from parturition to uterine involution or for days to first ovulation. Energy balance was less for the high group during wk 1, 2, 10, and 11. Plasma glucose concentration was lowest during wk 2 for both groups, and nonesterified fatty acids and beta-hydroxybutyrate were greatest for both groups during wk 1 and 2. Liver glycogen content was lower at d 15 postpartum for the high group, and liver triglyceride content was greater on d 30 for the high group. The data for reproductive functions support the concept that high milk production is antagonistic to expression of estrous behavior but not to reactivation of ovarian function.

Estrogen formation is catalyzed by the aromatase cytochrome P450 (P450_AROM) enzyme. Aromatase activity has been detected in several regions in the rat brain. In the present study, we used peptide-generated polyclonal antibodies raised against a 20-amino acid synthetic fragment of the rat P450_AROM protein (as deduced from the nucleic acid sequence of the rat P450_AROM complementary DNA), to determine the location of this enzyme in rat brain sections. Immunoreactive antisera were titered by means of an enzyme-linked immunosorbent assay and purified by diethylaminoethyl-Affigel Blue chromatography. Specific immunoreactivity was confirmed by Western blot analysis using known aromatase-containing tissue (rat ovary homogenates and microsomal fractions). Evaluation of the distribution of P450_AROM immunoreactivity in brain sections of male and female rats (30 and 60 days of age) was performed using the avidin biotin peroxidase immunocytochemical technique and light microscopy. P450_AROM immunoreactivity appeared to be localized to neurons, and was present in brain regions and nuclei where enzymatic activity has been reported. For example, intense immunoreactivity was observed in the amygdaloid structures and supraoptic nucleus, whereas moderate to light immunoreactivity was evident in the paraventricular and arcuate nuclei and hippocampus. Surprisingly, neurons in the bed nucleus stria terminalis, medial basal hypothalamic, and preoptic areas displayed little aromatase immunoreactivity. However, P450_AROM immunoreactivity was detected in specific brain regions not previously recognized to contain the enzyme (i.e. intense staining was seen in the reticular thalamic nucleus, olfactory tract and piriform cortex, as well as other brain structures). The pattern, distribution, and intensity of P450_AROM immunoreactivity was similar regardless of sex or age. In this study, microsomal preparations derived from a new brain area (i.e. the reticular thalamic nucleus; Rt) displaying P450_AROM immunoreactivity were observed to contain detectable levels of aromatase enzymatic activity, as determined by the 3H2O-release assay. The activity in the Rt was inhibited by a known aromatase inhibitor, 4-hydroxyandrostenedione. These results confirm histologically the localization of P450_AROM to brain regions where aromatase enzymatic activity has been detected and extend the knowledge of its location to areas previously unknown as sites of aromatase activity, which may be involved in the modulation of neuroendocrine function and reproductive behavior. (Endocrinology129: 2834-2844, 1991)

The objectives of this study were to develop a rapid method for sex determination for several mammalian species using polymerase chain reaction (PCR) and to use this method to determine whether there is a significant developmental difference in spherical diameter between male and female d-10 or -11 porcine embryos. The PCR system was developed and verified using genomic DNA from pigs of known sex, then it was tested with genomic DNA from several other mammalian species. Sex is determined by amplification of two genes in a single reaction. The presence or absence of a region of the Sry (sex-determining region Y) gene determines sex, and amplification of the Zfy (male) or Zfx (female) genes acts as a positive control for PCR. Sex determination was successful for all animals tested, including pigs, cattle, sheep, goats, llamas, horses, humans, baboons, dogs, cats, rats, and mice. A total of 209 embryos were collected from 21 crossbred gilts on d 10 or 11 of gestation, and their diameters were measured. No significant difference in embryo diameter was detected between male and female embryos, indicating that sexual dimorphism in embryonic growth in pigs does not occur before the period of rapid embryo elongation. The present sexing technique using PCR is rapid (approximately 6 h from receipt of embryos to results), and it may be useful for examining the effects of sex on any trait of interest in early porcine embryos and embryos from several other mammals.