TABLE 1.

Effects of different doses of ΔFSH-occludin conjugate on the BTB integrity and germ cell loss from the seminiferous epithelium in adult Sprague-Dawley rats

Treatment	ΔFSH-occludin conjugate* (protein/rat)	FSH (Mr 40 kDa) (Mr ~2200)	Occludin Peptide**	Effects on BTB***	Effects on germ cell loss***
Regimen 1	40 µg	1 nmol (40 µg)	3 nmol (~6.6 µg)	+	ns
Regimen 2	150 µg	3.5 nmol (140 µg)	10.5 nmol (~20 µg)	+	+

^*The protein concentration was estimated by Coomassie blue dye-binding assay using BSA as a standard and was administered to each rat (~300 gm b.w.) via i.p. (n=3 rats).

^**The mass of occludin containing in the FSH mutant-occludin peptide conjugate was estimated based on the stoichiometric ratio of FSH mutant:occludin peptide in the conjugate at ~1:3 (see Fig. 1). It is noted that the mass of occludin in the conjugate may be an underestimate since succinimidyl 4-formylbenzoate (SFB) also reacted with other primary amines found in Lys in addition to the N-terminus of the α and β subunit, yielding additional aldehyde groups that reacted with the hydrazine functional groups in the 22-amino acid peptide, forming the stable hydrazone conjugate (see Fig. 1).

^***This scoring is based on results of electron microscopy and fluorescent microscopy studies to assess BTB integrity, and/or histological analysis (to assess effects on germ cell loss) on rats sacrificed by week 9 after treatment. In Regimen 1, <10% of the seminiferous tubules were affected having tubules with signs of germ cell loss from the epithelium; in Regimen 2, ~30% of the tubules were affected having tubules with visible germ cell loss (e.g., elongating and round spermatids, and spermatocytes found in tubule lumen (see Fig. 5). About 600 tubules were examined and scored from three rats. ns, not statistically significant; +, detectable germ cell loss.