The red deer is a seasonally breeding mammal with a circannual cycle of prolactin secretion which reaches its peak during the non-breeding season. This study investigated expression of the prolactin receptor gene in red deer tissues collected in the breeding and non-breeding seasons. A 562 bp fragment of the extracellular domain of the red deer prolactin receptor cDNA was amplified from red deer liver poly(A)+ RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) using primers designed from the human sequence. Northern blots were prepared using 10-20 micrograms poly(A)+ RNA. The blots were hybridized to the 562 bp cDNA labelled by random priming with alpha 32P-dCTP. A main transcript of 3.5 kb was expressed in liver, heart, kidney and testis throughout the year and in epididymis during the breeding season only. In the testis an additional major transcript of 1.7 kb was present during the breeding and non-breeding seasons. Competitive binding assays using 125I-ovine prolactin (125I-oPRL) were performed on microsomal membrane fractions prepared from liver. Scatchard analyses confirmed the presence of a single class of lactogen-binding receptor with a mean Ka of 0.87 +/- 0.12 x 10(9) M-1 and a Bmax of 73.6 +/- 9.8 fmol/mg protein (n = 5). Cross-linking of 125I-oPRL to liver microsomes with 0.5 mM disuccinimidyl suberate followed by SDS-PAGE revealed a major band of molecular mass 56 kDa which was displaced by ovine prolactin, suggesting a specific lactogen-binding entity of 33 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)
Two experiments were conducted with mature Merino ewes to investigate the effects of time and site of insemination of fresh and frozen-thawed semen on the fertility of superovulated ewes. In Experiment 1, each ewe was treated with an intravaginal progestagen sponge and PMSG and/or FSH. They were inseminated in the uterus with fresh or frozen-thawed semen (approximately 100 x 10(6) motile sperm) at 24, 44 or 64 h after sponge withdrawal. Ova were recovered at 88 h after sponge withdrawal and classified as fertilized if they had pronuclei or had cleaved. Mean fertilization rates of recovered ova were 60.0, 93.7 and 87.8% for fresh semen and 46.1, 98.2 and 26.1% for frozen-thawed semen at each of the insemination times (24, 44 and 64 h) respectively. Overall, fertilization rates were higher for fresh semen than for frozen-thawed semen (P less than 0.01), but there was an interaction with time of insemination (P less than 0.01). Following insemination with frozen-thawed semen at 64 h, only 61% of the fertilized ova developed to the 2- to 4-cell stage by the time of embryo recovery; this was less than in any of the other groups (P less than 0.05). In Experiment 2, ewes were inseminated with 100 x 10(6) motile fresh or frozen-thawed semen in the uterus or in the oviducts at 64 h after sponge withdrawal.(ABSTRACT TRUNCATED AT 250 WORDS)
This study investigated the pattern and site of expression of the prolactin receptor gene in the testis and epididymis of red deer collected during the breeding season (n=3). Ribonuclease protection assays using 50 µg total RNA and a 300 bp [ 32 P]-labelled antisense cRNA probe, generated from the extracellular domain of the red deer prolactin receptor, confirmed the expression of the receptor in both the testis and epididymis; a higher level of prolactin receptor mRNA was detected in the epididymis compared with the testis (170·4 1·5 10 3 and 26·3 2·7 10 3 arbitrary units respectively; P<0·05). In situ hybridisation using 300 bp [33 P]-labelled sense and antisense cRNA probes generated from the extracellular domain of the receptor localised the expression sites to the seminiferous tubules and interstitial compartments of the testis and the epithelial layer of the epididymal duct. Quantification of grain numbers demonstrated a higher level of expression of the receptor in the epididymis compared with the interstitial and seminiferous tubule compartments of the testis (18·1 4·4 10 2 , 10·1 2·0 10 2 and 8·3 0·8 10 2 grains/µm 2 respectively; P<0·05). However, no differences were detected in the level of expression of the receptor between the interstitial and seminiferous tubule compartments of the testis. Immunocytochemistry using an anti-prolactin receptor antibody, raised against a peptide sequence from the extracellular domain of the rat prolactin receptor, localised expression of the receptor gene to the Leydig cells, pachytene spermatocytes, round spermatids and elongating spermatids. In the epididymis, the receptor was localised to the epithelial layer within the epididymal ducts. Expression of the prolactin receptor gene in the red deer testis and epididymis suggests a role for the hormone in steroidogenesis and spermatogenesis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.