BRIEF REPORT

The Use of Counterflow Centrifugation to Enrich Gonadotropes and Somatotropes

Correspondence to: Gwen V. Childs, Dept. of Anatomy, Univ. of Arkansas for Medical Sciences, 4301 West Markham Street, Slot 510, Little Rock, AR 72205-7199.

	Summary

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Counterflow centrifugation produces populations of gonadotropes or growth hormone (GH) cells enriched to 90% in a Beckman elutriator. The pituitary populations are first separated by size into three fractions applying different flow rates, stimulated with either gonadotropin-releasing hormone (GnRH) to enlarge the gonadotropes or growth hormone-releasing hormone (GHRH) to enlarge the somatotropes for 3 hr. The fractions are re-eluted, first at the original flow rates and then at higher flow rates to separate enlarged gonadotropes or somatotropes. Most other cell types are reduced to less than 5%. However, co-storage of GH and gonadotropin antigens is seen in either population. Enriched gonadotropes or somatotropes can be used in studies of proliferation, autocrine or paracrine regulation, or ion channel functions.

(J Histochem Cytochem 49:663–664, 2001)

Key Words: counterflow centrifugation, gonadotropes

	Introduction

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The anterior pituitary consists of a mixture of hormone-producing cells that can be differentiated cytochemically by the type of hormone produced (Moriarty 1973 ; Childs et al. 1983 ). Studies of responses and functions of cell types have used tumor cells, purified them (Childs et al. 1988 , Childs et al. 1992 ; Vankelecom and Denef 1997 ), or provided identifying markers (Childs and Burke 1987 ; Childs et al. 1987 ; Kineman et al. 1990 ).Our laboratory developed a method that produced a ninefold enrichment of pituitary corticotropes (Childs et al. 1988 ). More recently, we have worked on techniques that enrich gonadotropes or somatotropes. Male or female Sprague–Dawley rats are acclimated 7–10 days before use, as approved by committees at both University of Texas Medical Branch and University of Arkansas for Medical Science. During the dispersion, single-cell preparations are obtained (Childs et al. 1988 , Childs et al. 1994 , Childs et al. 2000 ). Pituitaries from four to six rats produce 10 million pituitary cells and 1 million somatotropes or gonadotropes.

The Beckman elutriator rotor is assembled with the Sanderson chamber, which allows work with small numbers (10,000–10,000,000) of cells. In the centrifuge, the rotor is attached to tubing and a peristaltic pump that delivers fluid into the chamber at regulated flow rates. Initially, 500 ml of 70% ethanol is run through the rotor at about 10 ml/min for sterilization while the centrifuge is off. Cold sterile water (on ice) is then run through the chamber, removing bubbles, followed by 200 ml Dulbecco's PBS with gentamyacin and bovine serum albumin (BSA). The centrifuge is then closed and run at 1960 rpm. This tests for leaks and bubbles in the system that must be corrected.

Single-cell dispersions are then loaded at 8–10 ml/min as 50 ml is collected (on ice) and the centrifuge is running at 1960 rpm. They are loaded through a syringe connected to the tubing, just before the pump. This allows the cells to settle into the Sanderson chamber, with the largest cells at the bottom and layers of smaller cells towards the top of the pellet. Then the flow rate is increased to collect larger cells sequentially in each of three fractions (Fr) Fr 1, 2, and 3 (50 ml each) are collected at 15, 25, and 35 ml/min, respectively. As Fr 3 is collected, the centrifuge is stopped, excess fluid in the tubing collected, the rotor removed, and chamber contents added. After reassembly, the rotor is prepared for the second elutriation by pumping more PBS through and eliminating bubbles and leaks. It is maintained at 0–4C until re-elutriation.

The three fractions are spun down at 900 rpm and pellets are resuspended in Dulbecco's modified Eagle's medium (high-glucose DMEM) including 2.5 µg/500 ml HEPES buffer, 0.3% BSA, 5 µg/ml insulin, 30 nM sodium selenite, 50 µg/ml transferrin, and 4.2 µg/ml fibronectin. The load fraction is added to Fr 1. After stimulation with GnRH (for gonadotropes) or GHRH (for somatotropes) for 3 hr at 37C, Fr 1, 2, and 3 are re-eluted separately. Each is loaded at 10 ml/min (50 ml is collected) and re-eluted first at the original flow rate (Fr 1, 15 ml/min; Fr 2, 25 ml/min; Fr 3, 35 ml/min) and then at a new flow rate to collect larger cells (5 ml/min higher for somatotropes or 10 ml/min higher for gonadotropes.)

Samples taken from the original and enlarged fractions were immunolabeled. For example, before re-elutriation, Fr 1, 2, and 3 contained 40 ± 8%, 45 ± 0.2%, and 36 ± 10% GH cells, respectively. After stimulation and re-elutriation, enlarged cells from these same fractions contained 84 ± 4%, 95 ± 0.3%, and 91 ± 2% GH cells, respectively. Pooled fractions from more than five or six somatotrope enrichments contain an average of 90 ± 2% GH cells. Similarly, pooled fractions of enriched gonadotropes contain 92 ± 3% cells with gonadotropins.

Most populations contain <5% other cell types. The storage overlap between GH and gonadotropins persists (Childs et al. 1994 , Childs et al. 2000 ). At least 15% of GH cells contain gonadotropins and 15–30% of the enriched gonadotropes contain GH. Fig 1 shows immunolabeling for LH and FSH in enriched gonadotropes, with the label in various shades of gray. Enriched gonadotropes immunolabeled for prolactin (Fig 1C), ACTH (Fig 1D), or GH (Fig 1E) have few cells.

View larger version (108K): [in this window] [in a new window]   Figure 1. Immunolabeling for LH and FSH in enriched gonadotropes (A, B). Bars = 20 µm. Enriched gonadotropes labeled for prolactin (C), ACTH (D), or GH (E) have few cells. Arrow in E, GH cell. Bars = 10 µm.

We run a two-step elutriation process because the smallest gonadotropes or somatotropes do not enlarge sufficiently to be separated from the largest cells in the pituitary. This protocol can separate cells that differ in area by 5–10 µm². Small cells can easily enlarge by that amount. However they can be separated only if they are first eluted with their small cell counterparts. Although this technique does not give yields suitable for purifications or biochemical assays, it produces a >90% enrichment, uses few animals, and can be completed in one full day. It is useful in studies of proliferation, ion channel activity, RT-PCR, or cell communication.

	Footnotes

Presented in part at the Joint Meeting of the Histochemical Society and the International Society for Analytical and Molecular Morphology, Santa Fe, NM, February 2–7, 2001.

Received for publication December 19, 2000; accepted February 16, 2001.

	Literature Cited

Top Summary Introduction Literature Cited

Childs GV, Burke J (1987) Use of the reverse hemolytic plaque assay to study the regulation of anterior lobe ACTH secretion by CRF, AVP, A-II and glucocorticoids. Endocrinology 120:439-444[Abstract]

Childs GV, Lloyd JM, Unabia G, Rougeau D (1988) Enrichment of corticotropes by counterflow centrifugation. Endocrinology 123:2885-2895[Abstract]

Childs GV, Naor Z, Hazum E, Tibolt R, Westlund KN, Hancock MB (1983) Cytochemical characterization of pituitary target cells for biotinylated gonadotropin releasing hormone. Peptides 4:549-555[Medline]

Childs GV, Unabia G, Burke JA, Marchetti C (1987) Secretion from corticotropes after avidin-fluorescein stains for biotinylated ligands (CRF or AVP). Am J Physiol 252:E347-356[Abstract/Free Full Text]

Childs GV, Unabia G, Lee BL, Rougeau D (1992) Heightened secretion by small and medium-sized luteinizing hormone (LH) gonadotropes late in the cycle suggests contributions to the LH surge or possible paracrine interactions. Endocrinology 130:345-352[Abstract]

Childs GV, Unabia G, Miller BT (1994) Cytochemical detection of GnRH binding sites on rat pituitary cells with LH, FSH and GH antigens during diestrous upregulation. Endocrinology 134:1943-1956[Abstract]

Childs GV, Unabia G, Wu P (2000) Differential expression of growth hormone messenger ribonucleic acid by somatotropes and gonadotropes in male and cycling female rats. Endocrinology 141:1560-1570[Abstract/Free Full Text]

Kineman RD, Faught WJ, Frawley LS (1990) Bovine pituitary cells exhibit a unique form of somatotrope secretory heterogeneity. Endocrinology 127:2229-2235[Abstract]

Moriarty GC (1973) Adenohypophysis: ultrastructural cytochemistry. A review. J Histochem Cytochem 21:855-892[Medline]

Vankelecom H, Denef C (1997) Paracrine communication in the anterior pituitary as studied in reaggregate cell cultures. Microsc Res Tech 39:150-156[Medline]

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